Fgf21 mimetic antibodies and uses thereof

ABSTRACT

The present disclosure relates to monoclonal antibodies and antigen-binding fragments thereof that bind to human β-klotho, and pharmaceutical compositions and methods of treatment comprising the same.

This application is a division of U.S. patent application Ser. No.17/127,600, filed Dec. 18, 2020, now U.S. Pat. No. 11,692,046, which isa division of U.S. patent application Ser. No. 15/890,302, filed Feb. 6,2018, now U.S. Pat. No. 10,899,844, which claims the benefit of U.S.Provisional Application No. 62/456,609 filed on Feb. 8, 2017, which ishereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on May 30, 2023, isnamed 51571-003004_SL.xml and is 85,547 bytes in size.

FIELD

The present disclosure relates to fibroblast growth factor 21 (FGF21)mimetic antibodies. Also disclosed are methods for treatingFGF21-associated disorders, such as obesity, type 1 and type 2 diabetesmellitus, pancreatitis, dyslipidemia, nonalcoholic steatohepatitis(NASH), insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, metabolic syndrome, and other metabolic disorders, and inreducing the mortality and morbidity of critically ill patients.

BACKGROUND

The fibroblast growth factor (FGF) family is characterized by 22genetically distinct, homologous ligands, which are grouped into sevensubfamilies. According to the published literature, the FGF family nowconsists of at least twenty-three members, FGF-1 to FGF-23 (Reuss et al.(2003) Cell Tissue Res. 313:139-157).

Fibroblast growth factor 21 (FGF21) was isolated from mouse embryos andis closest to FGF19 and FGF23. This FGF subfamily regulates diversephysiological processes uncommon to classical FGFs, namely energy andbile acid homeostasis, glucose and lipid metabolism, and phosphate aswell as vitamin D homeostasis. Moreover, unlike classical FGFs, thissubfamily acts in an endocrine fashion (Moore, D. D. (2007) Science 316,1436-8). FGF21 has been reported to be preferentially expressed in theliver (Nishimura et al. (2000) Biochimica et Biophysica Acta,1492:203-206; patent publication WO01/36640; and patent publicationWO01/18172) and described as a treatment for ischemic vascular disease,wound healing, and diseases associated with loss of pulmonary, bronchiaor alveolar cell function and numerous other disorders.

FGF21 has been identified as a potent metabolic regulator. Systemicadministration of FGF21 to rodents and rhesus monkeys with diet-inducedor genetic obesity and diabetes exerts strong anti-hyperglycemic andtriglyceride-lowering effects, and reduction of body weight (Coskun, T,et al. (2008) Endocrinology 149:6018-6027; Kharitonenkov, A. et al.(2005) Journal of Clinical Investigation 115:1627-1635; Kharitonenkov,A., et al. (2007) Endocrinology 148:774-781; Xu, J, et al. (2009)Diabetes 58:250-259). FGF21 is a 209 amino acid polypeptide containing a28 amino acid leader sequence. Human FGF21 has about 79% amino acididentity to mouse FGF21 and about 80% amino acid identity to rat FGF21.

In mammals, FGFs mediate their action via a set of four FGF receptorsFGFR1-4 that in turn are expressed in multiple spliced variants. EachFGF receptor contains an intracellular tyrosine kinase domain that isactivated upon ligand binding, leading to downstream signaling pathwaysinvolving MAPKs (Erk1/2), RAF1, AKT1 and STATs. (Kharitonenkov, A. etal. (2008) BioDrugs 22:37-44). Several reports suggested that the“c”-reporter splice variants of FGFR1-3 exhibit specific affinity to3-klotho and could act as endogenous receptors for FGF21 (Kurosu et al.,2007 J. Biol. Chem. 282:26687-26695); Ogawa et al., 2007 Proc. Natl.Acad. Sci. USA 104:7432-7437; Kharitonenkov et al., 2008 J. CellPhysiol. 215, 1-7). In 3T3-L1 cells and white adipose tissue, FGFR1 isby far the most abundant receptor, and it is therefore most likely thatFGF21's main functional receptors in this tissue are the β-klotho-FGFR1ccomplexes.

Although FGF21 activates FGF receptors and downstream signalingmolecules, including FRS2a and extracellular signal-regulated kinase(ERK), direct interaction of FGFRs and FGF21 has not been detected.Furthermore, various non-adipocyte cells do not respond to FGF21, eventhough they express multiple FGFR isoforms. All of these data suggestthat a cofactor must mediate FGF21 signaling through FGFRs. Studies haveidentified beta-klotho (β-klotho), which is highly expressed in liver,adipocytes and in pancreas, as a determinant of the cellular response toFGF21 (Kurosu, H. et al. (2007) J Biol Chem 282, 26687-95). Theβ-klotho-FGFR complex, but not FGFR alone, binds to FGF21 in vitro(Kharitonenkov, A., et al. (2008) J Cell Physiol 215, 1-7). FGF21 bindsto β-klotho in complex with FGFR1c, 2c, or 3c; but not to β-klotho incomplex with FGFR4 (Owen et al., 2015 Trends in Endocrinology 26:22-29). A similar mechanism has been identified in the FGF23-klotho-FGFRsystem (Urakawa, I. et al. (2006) Nature 444, 770-4).

The bioactivity of FGF21 was first identified in a mouse 3T3-L1adipocyte glucose uptake assay (Kharitonenkov, A. et al. (2005) J ClinInvest 115, 1627-35). Subsequently, FGF21 was shown to induceinsulin-independent glucose uptake and GLUT1 expression. FGF21 has alsobeen shown to ameliorate hyperglycemia in a range of diabetic rodentmodels. In addition, transgenic mice over-expressing FGF21 were found tobe resistant to diet-induced metabolic abnormalities, includingdecreased body weight and fat mass, and enhancements in insulinsensitivity (Badman, M. K. et al. (2007) Cell Metab 5, 426-37).Administration of FGF21 to diabetic non-human primates (NHP) caused adecline in fasting plasma glucose, triglycerides, insulin and glucagonlevels, and led to significant improvements in lipoprotein profilesincluding a nearly 80% increase in HDL cholesterol (Kharitonenkov, A. etal. (2007) Endocrinology 148, 774-81). Importantly, hypoglycemia was notobserved at any point during this NHP study. Other studies identifiedFGF21 as an important endocrine hormone that helps to control adaptationto the fasting state. This provides a previously missing link,downstream of PPARα, by which the liver communicates with the rest ofthe body in regulating the biology of energy homeostasis. The combinedobservations that FGF21 regulates adipose (lipolysis), liver (fatty acidoxidation and ketogenesis), and brain (torpor) establish it as a majorendocrine regulator of the response to fasting (Kharitonenkov, A. &Shanafelt, A. B. (2008) BioDrugs 22, 37-44).

The problem with using FGF21 directly as a biotherapeutic is that itshalf-life is very short. In mice, the half-life of human FGF21 is 0.5 to1 hours, and in cynomolgus monkeys, the half-life is 2 to 3 hours.Furthermore, when wild type FGF21 is used in pharmaceutical formulationsor preparations, its stability is adversely affected by preservativese.g., m-cresol.

SUMMARY

The present disclosure relates to FGF21 mimetic antibodies, i.e.,monoclonal antibodies that bind to beta-klotho (β-klotho) and activatethe human Fibroblast Growth Factor 21 (hereinafter, sometimes referredto as “FGF21”) receptor complex and FGF21-mediated signaling (e.g.,FGF21-receptor-dependent signaling), antigen-binding fragments thereof,and pharmaceutical compositions and methods of treatment comprising thesame.

In specific aspects, antigen-binding fragments (of the FGF21 mimetic,(β-klotho-binding antibodies) of the disclosure can be molecules withFGF21-like activity and selectivity but with added therapeuticallydesirable characteristics such as protein stability, low immunogenicity,ease of production and a desirable in vivo half-life.

The monoclonal FGF21 mimetic antibodies of the present disclosure,antigen-binding fragments thereof, and pharmaceutical compositionscomprising the same are useful for the treatment of FGF21-associateddisorders, such as obesity, type 2 diabetes mellitus, type 1 diabetesmellitus, pancreatitis, dyslipidemia, nonalcoholic steatohepatitis(NASH), insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease,atherosclerosis, peripheral arterial disease, stroke, heart failure,coronary heart disease, kidney disease, diabetic complications,neuropathy, gastroparesis and other metabolic disorders, and in reducingthe mortality and morbidity of critically ill patients.

In particular aspects, isolated FGF21 mimetic antibodies, orantigen-binding fragments, described herein bind β-klotho, with anequilibrium dissociation constant (K_(D)) of less than or equal to 500pM or 400 pM, for example as determined by BIACORE™ binding assay, andmay also activate the cynomolgus monkey FGFR1c_β-klotho receptor complexwith an EC50 of less than or equal to 50 nM, for example as measured byextracellular signal-regulated kinase (ERK) phosphorylation (pERK orphospho-ERK) cell assays. In particular aspects, isolated FGF21 mimeticantibodies, or antigen-binding fragments, described herein bindβ-klotho, with an equilibrium dissociation constant (K_(D)) of less thanor equal to 300 pM or 400 pM, for example as determined by BIACORE™binding assay, and may also activate the cynomolgus monkeyFGFR1c_β-klotho receptor complex with an EC50 of less than or equal to50 nM, for example as measured by pERK cell assays.

In particular aspects, isolated FGF21 mimetic antibodies, orantigen-binding fragments, described herein bind β-klotho, with anequilibrium dissociation constant (K_(D)) of less than or equal to 100pM or 50 pM. For example, isolated antibodies or antigen-bindingfragments described herein may bind to human β-klotho with a K_(D) ofless than or equal to 100 pM, less than or equal to 50 pM, less than orequal to 45 pM, less than or equal to 40 pM, less than or equal to 35pM, less than or equal to 25 pM, or less than or equal to 15 pM. Morespecifically, the isolated antibodies or antigen-binding fragmentsdescribed herein may also bind human β-klotho with a K_(D) of less thanor equal to 10 pM, as measured by BIACORE™ binding assay or solutionequilibrium titration assay (SET); and may also activate the cynomolgusmonkey FGFR1c_β-klotho receptor complex with an EC50 of less than orequal to 50 nM, for example as measured by pERK cell assays.

The present disclosure relates to an isolated antibody, orantigen-binding fragments thereof, that binds to human and cynomolgusmonkey β-klotho. The present disclosure also relates to an isolatedantibody, or antigen-binding fragments thereof, that binds β-klotho andactivates the FGF21 receptor complex and FGF21-mediated signaling (e.g.,FGF21-receptor-dependent signaling). In particular aspects, an isolatedantibody or antigen-binding fragment thereof described herein does notactivate human FGFR2c_β-klotho, FGFR3c_β-klotho, or FGFR4 β-klothoreceptor complexes.

The present disclosure also relates to an isolated antibody, orantigen-binding fragments thereof, that binds β-klotho and furthercompetes for binding with an antibody as described in Table 1, forexample, antibody NOV005 or NOV006. The present disclosure also furtherrelates to an isolated antibody, or antigen-binding fragments thereof,that binds the same epitope as an antibody as described in Table 1, forexample, antibody NOV005 or NOV006.

As described here, “competition” between antibodies and/orantigen-binding fragments thereof signifies that both antibodies (orbinding fragments thereof) bind to the same β-klotho epitope (e.g., asdetermined by a competitive binding assay, by any of the methods wellknown to those of skill in the art). An antibody or antigen-bindingfragment thereof also “competes” with a β-klotho antibody orantigen-binding fragment of the present disclosure (e.g., NOV005 orNOV006) if said competing antibody or antigen-binding fragment thereofbinds the same β-klotho epitope, or an overlapping β-klotho epitope, asan antibody or antigen-binding fragment of the present disclosure. Asused herein, a competing antibody or antigen-binding fragment thereofcan also include one which (i) sterically blocks an antibody orantigen-binding fragment of the present disclosure from binding itstarget (e.g., if said competing antibody binds to a nearby,non-overlapping β-klotho and/or (β-klotho epitope and physicallyprevents an antibody or antigen-binding fragment of the presentdisclosure from binding its target); and/or (ii) binds to a different,non-overlapping β-klotho epitope and induces a conformational change tothe β-klotho protein such that said protein can no longer be bound by aβ-klotho antibody or antigen-binding fragment of the present disclosurein a way that would occur absent said conformational change.

The binding affinity of isolated antibodies and antigen-bindingfragments described herein can be determined by solution equilibriumtitration (SET). Methods for SET are known in the art and are describedin further detail below. Alternatively, binding affinity of the isolatedantibodies, or fragments, described herein can be determined by Biacoreassay. Methods for Biacore kinetic assays are know in the art and aredescribed in further detail below.

The isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, may be used to increase the activation of the FGF21 receptorcomplex, and thereby, the FGF21 signaling pathway. In a particularaspect, isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, may be used to increase the activation of the FGF21 receptorcomplex, and thereby, the FGF21 signaling pathway, by at least about15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%.

The isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, as described herein can be monoclonal antibodies, human orhumanized antibodies, chimeric antibodies, single chain antibodies, Fabfragments, Fv fragments, F(ab′)2 fragments, or scFv fragments, and/orIgG isotypes (e.g., IgG1, IgG2, or IgG4).

The isolated FGF21 mimetic antibodies, or antigen-binding fragmentsthereof, as described herein can also include a framework in which anamino acid has been substituted into the antibody framework from therespective human VH or VL germline sequences.

Another aspect of the present disclosure includes an isolated antibodyor antigen-binding fragments thereof having the full heavy and lightchain sequences of Fabs described in Table 1, for example, antibodyNOV005 or NOV006. More specifically, the isolated antibody orantigen-binding fragments thereof can have the heavy and light chainsequences of Fab NOV005 or NOV006.

A further aspect of the present disclosure includes an isolated antibodyor antigen-binding fragments thereof comprising the heavy and lightchain variable domain sequences of Fabs described in Table 1, forexample NOV005 or NOV006. More specifically, the isolated antibody orantigen-binding fragment thereof comprises the heavy and light chainvariable domain sequence of Fab NOV005 or NOV006.

The present disclosure also relates to compositions (e.g.,pharmaceutical compositions) comprising an isolated antibody, orantigen-binding fragments thereof, described herein (e.g., NOV005 orNOV006), as well as, antibody compositions in combination with apharmaceutically acceptable carrier. Specifically, the presentdisclosure further includes pharmaceutical compositions comprising anantibody or antigen-binding fragments thereof of Table 1, such as, forexample antibody NOV005 or NOV006. The present disclosure also relatesto pharmaceutical compositions comprising a combination of two or moreof the isolated antibodies or antigen-binding fragments thereof of Table1, for example, antibody NOV005 or NOV006.

The present disclosure also relates to an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding the heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 15. Inparticular aspects, the nucleic acid molecule comprises a sequence thathas at least 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID Nos: 16, 36, or 38. In a further aspect of thepresent disclosure, a nucleic acid molecule provided herein comprisesthe nucleic acid sequence of SEQ ID NO: 16, 36, or 38.

The present disclosure also relates to an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding a light chain variableregion having an amino acid sequence of SEQ ID NO: 26 or 32. Inparticular aspects, the nucleic acid molecule comprises a sequence thathas at least 90% sequence identity to a nucleic acid sequence of SEQ IDNO: 27, 54, 33, or 40. In a further aspect of the present disclosure anucleic acid molecule provided herein comprises the nucleic acidsequence of SEQ ID NO: 27, 54, 33, or 40.

The present disclosure also relates to a vector that includes one ormore of the nucleic acid molecules described herein. In specificaspects, a first vector encodes a heavy chain variable region or heavychain of an antibody provided herein, such as NOV005 or NOV006, a secondvector encodes a light chain variable region or heavy chain of anantibody provided herein, such as NOV005 or NOV006. The first vector andsecond vector are transduced into a host cell for coexpression to formantibodies comprising such heavy chains and such light chains.

The present disclosure also relates to an isolated host cell thatincludes a recombinant DNA sequence encoding a heavy chain of theantibody described above, and a second recombinant DNA sequence encodinga light chain of the antibody described above, wherein said DNAsequences are operably linked to a promoter and are capable of beingexpressed in the host cell. It is contemplated that the antibody can bea human monoclonal antibody. It is also contemplated that the host cellis a non-human mammalian cell, for example a CHO cell or HEK293 cell.

The present disclosure also relates to activating a Fibroblast GrowthFactor 21 (FGF21) receptor, and, thereby, FGF21-mediated signaling(e.g., FGF21-receptor-dependent signaling), wherein the method includesthe step of contacting a cell with an effective amount of a compositioncomprising the isolated antibody or antigen-binding fragments thereofdescribed herein.

In one particular aspect, it is contemplated that the cell is a humancell. It is further contemplated that the cell is in a subject. In oneembodiment, it is contemplated that the cell is an adipocyte. In otherembodiments, the cell may be one or more of hepatocytes, pancreas cells,endothelial cells, muscle, or renal cells. In specific aspects, it isstill further contemplated that the subject is human.

The present disclosure also relates to a method of treating, managing,improving, or preventing a FGF21-associated disorder in a subject,wherein the method includes the step of administering to the subject aneffective amount of a composition comprising the antibody orantigen-binding fragments thereof described herein (e.g., NOV005 orNOV006). In one aspect, the FGF21-associated disorder is obesity. In oneaspect, the FGF21-associated disorder is type 2 diabetes. It iscontemplated that the subject is human.

Any of the foregoing isolated antibodies or antigen-binding fragmentsthereof may be a monoclonal antibody or antigen-binding fragmentsthereof.

Non-limiting embodiments of the disclosure are described in thefollowing aspects:

1. An isolated antibody or antigen-binding fragment thereof that bindsto an epitope of β-klotho, wherein the antibody or antigen-bindingfragment thereof comprises:

-   -   a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of        SEQ ID NO: 6, 9, 10 or 12;    -   a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of        SEQ ID NO: 7, 11 or 13;    -   a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of        SEQ ID NO: 8 or 14;    -   a light chain CDR1 (LCDR1) comprising the amino acid sequence of        SEQ ID NO: 19, 31, 22, or 25;    -   a light chain CDR2 (LCDR2) comprising the amino acid sequence of        SEQ ID NO: 20 or 23; and    -   a light chain CDR3 (LCDR3) comprising the amino acid sequence of        SEQ ID NO: 21 or 24.

2. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises:

-   -   a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of        SEQ ID NO: 6, 9, 10 or 12;    -   a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of        SEQ ID NO: 7, 11 or 13; a heavy chain CDR3 (HCDR3) comprising        the amino acid sequence of SEQ ID NO: 8 or 14;    -   a light chain CDR1 (LCDR1) comprising the amino acid sequence of        SEQ ID NO: 31, 22, or 25;    -   a light chain CDR2 (LCDR2) comprising the amino acid sequence of        SEQ ID NO: 20 or 23; and    -   a light chain CDR3 (LCDR3) comprising the amino acid sequence of        SEQ ID NO: 21 or 24.

3. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises:

-   -   a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of        SEQ ID NO: 6, 9, 10 or 12;    -   a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of        SEQ ID NO: 7, 11 or 13;    -   a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of        SEQ ID NO: 8 or 14;    -   a light chain CDR1 (LCDR1) comprising the amino acid sequence of        SEQ ID NO: 19, 31, 22, or 25;    -   a light chain CDR2 (LCDR2) comprising the amino acid sequence of        SEQ ID NO: 20 or 23; and    -   a light chain CDR3 (LCDR3) comprising the amino acid sequence of        SEQ ID NO: 21 or 24.

4. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises:

-   -   (i) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 6,        a HCDR2 comprising the amino acid sequence of SEQ ID NO: 7, a        HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 21;    -   (ii) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 9,        a HCDR2 comprising the amino acid sequence of SEQ ID NO: 7, a        HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 31, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 21;    -   (iii) a HCDR1 comprising the amino acid sequence of SEQ ID NO:        10, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11,        a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or    -   (iv) a HCDR1 comprising the amino acid sequence of SEQ ID NO:        12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 13,        a HCDR3 comprising the amino acid sequence of SEQ ID NO: 14, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 21.

5. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises:

-   -   (i) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 6,        a HCDR2 comprising the amino acid sequence of SEQ ID NO: 7, a        HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 21;    -   (ii) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 9,        a HCDR2 comprising the amino acid sequence of SEQ ID NO: 7, a        HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 21;    -   (iii) a HCDR1 comprising the amino acid sequence of SEQ ID NO:        10, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11,        a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or    -   (iv) a HCDR1 comprising the amino acid sequence of SEQ ID NO:        12, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 13,        a HCDR3 comprising the amino acid sequence of SEQ ID NO: 14, a        LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a        LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a        LCDR3 comprising the amino acid sequence of SEQ ID NO: 21.

6. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, aHCDR2 comprising the amino acid sequence of SEQ ID NO: 7, a HCDR3comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprisingthe amino acid sequence of SEQ ID NO: 19 or 31, a LCDR2 comprising theamino acid sequence of SEQ ID NO: 20, and a LCDR3 comprising the aminoacid sequence of SEQ ID NO: 21.

7. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, aHCDR2 comprising the amino acid sequence of SEQ ID NO: 7, a HCDR3comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprisingthe amino acid sequence of SEQ ID NO: 19 or 31, a LCDR2 comprising theamino acid sequence of SEQ ID NO: 20, and a LCDR3 comprising the aminoacid sequence of SEQ ID NO: 21.

8. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 10,a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, a HCDR3comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprisingthe amino acid sequence of SEQ ID NO: 22, a LCDR2 comprising the aminoacid sequence of SEQ ID NO: 23, and a LCDR3 comprising the amino acidsequence of SEQ ID NO: 24.

9. The isolated antibody or antigen-binding fragment thereof accordingto aspect 1, wherein the antibody or antigen-binding fragment thereofcomprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 12,a HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR3comprising the amino acid sequence of SEQ ID NO: 14, a LCDR1 comprisingthe amino acid sequence of SEQ ID NO: 25, a LCDR2 comprising the aminoacid sequence of SEQ ID NO: 23, and a LCDR3 comprising the amino acidsequence of SEQ ID NO: 21.

10. The antibody or antigen-binding fragment thereof according to anyone of aspects 1-9, wherein said antibody or fragment increases theactivity of β-klotho and FGFR1 c.

11. The antibody or antigen-binding fragment thereof according to anyone of aspect 1-9, which binds to a human β-klotho protein with a K_(D)of less than or equal to 450 pM, as measured by BIACORE™ binding assay.

12. The isolated antibody or antigen-binding fragment thereof accordingto any one of aspects 1-9, wherein said epitope comprises, or consistsessentially of, (i) one or more amino acids of residues 246-265,536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ ID NO:52); or(ii) one, two, three, four, five, or more amino acid residues from eachof the following stretches of residues 246-265, 536-550, 834-857 and959-986 of the β-klotho sequence (SEQ ID NO:52).

13. The isolated antibody or antigen-binding fragment thereof accordingto any one of aspects 1-9, wherein said epitope comprises, or consistsessentially of, (i) one or more of amino acids of residues 646-670,696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:52); or (ii)one, two, three, four, five, or more amino acid residues from each ofthe following stretches of residues 646-670, 696-700, and 646-689 of theβ-klotho sequence (SEQ ID NO:52).

14. The isolated antibody or antigen-binding fragment thereof accordingto any one of aspects 1-13, which is capable of activating thecynomolgus monkey FGFR1c-β-klotho receptor complex with an EC50 of lessthan or equal to 50 nM, as measured by pERK cell assays.

15. The isolated antibody or antigen-binding fragment of any one ofaspects 1-14, wherein said antibody or fragment does not contactresidues 701 (Tyr) or 703 (Arg) of human β-klotho (SEQ ID NO: 52).

16. The isolated antibody or antigen-binding fragment of any one ofaspects 1-15, wherein the antibody or fragment comprises a heavy chainvariable region (VH) comprising the amino acid sequence of SEQ ID NO: 15or an amino acid sequence with at least 90% or 95% identity thereof; anda light chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 26 or 32 or an amino acid sequence with at least 90% or 95%identity thereof

17. The isolated antibody or antigen-binding fragment of any one ofaspects 1-16, wherein the antibody or fragment comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 15.

18. The isolated antibody or antigen-binding fragment of any one ofaspects 1-17, wherein the antibody or fragment comprises a VL comprisingthe amino acid sequence of SEQ ID NO: 26 or 32.

19. The isolated antibody or antigen-binding fragment of aspect 17,wherein the antibody or fragment comprises a (i) a VH comprising theamino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acidsequence of SEQ ID NO: 26 or (ii) a VH comprising the amino acidsequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence ofSEQ ID NO: 32.

20. The isolated antibody or antigen-binding fragment of any one ofaspects 1-19, wherein the antibody comprises (i) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 17 and a light chaincomprising the amino acid sequence of SEQ ID NO: 28, or (ii) a heavychain comprising the amino acid sequence of SEQ ID NO: 17 and a lightchain comprising the amino acid sequence of SEQ ID NO: 34.

21. An isolated antibody or antigen-binding fragment thereof, whereinthe antibody or fragment binds to the same epitope as an isolatedantibody or fragment according to any one of aspects 1-20, wherein theantibody or antigen-binding fragment does not comprise (i) Combined orKabat CDRs of antibody NOV004 as set forth in Table 2; and/or (ii) aheavy chain variable domain comprising the amino acid sequence of SEQ IDNO: 43 or 55 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 47 or 57.

22. An isolated antibody or antigen-binding fragment thereof, whereinthe antibody or fragment competes for binding to β-klotho with anisolated antibody or fragment according to any one of aspects 1-20,wherein the antibody or antigen-binding fragment does not comprise (i)Combined or Kabat CDRs of antibody NOV004 as set forth in Table 2;and/or (ii) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO: 43 or 55 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 47 or 57.

23. The isolated antibody or antigen-binding fragment of any one ofaspects 1-20, wherein the antibody or fragment does not comprise (i)Combined or Kabat CDRs of antibody NOV004 as set forth in Table 2;and/or (ii) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO: 43 or 55 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 47 or 57.

24. A pharmaceutical composition comprising an antibody orantigen-binding fragment thereof of one of the above aspects and apharmaceutically acceptable carrier.

25. A method of treating a metabolic disorder comprising administeringto a subject afflicted with a metabolic disorder an effective amount ofa pharmaceutical composition comprising an antibody or antigen-bindingfragment according to any one of aspects 1-23.

26. The method of aspect 25, wherein the subject is afflicted with oneor more of obesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic steatohepatitis (NASH), insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia,hypertriglyceridemia, and metabolic syndrome.

27. The method of aspect 25, wherein the subject is afflicted with oneor more of obesity, diabetes, and dyslipidemia.

28. A method of treating a cardiovascular disorder comprisingadministering to a subject afflicted with a cardiovascular disorder aneffective amount of a pharmaceutical composition comprising an antibodyor fragment according to any one of the previous aspects.

29. The method of aspect 28, wherein the subject is afflicted with oneor more of atherosclerosis, peripheral arterial disease, stroke, heartfailure, and coronary heart disease.

30. An antibody or antigen-binding fragment thereof according to any oneof aspects 1-23, for use as a medicament.

31. A method of reducing body weight comprising administering to asubject in need thereof an effective amount of a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment accordingto any one of aspects 1-23.

32. A method of reducing appetite or food intake comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment according to any one of aspects 1-23.

33. A method of reducing plasma triglyceride (TG) concentrations orplasma total cholesterol (TC) concentrations in a subject, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment according to any one of aspects 1-23.

34. The method of aspect 31, 32, or 33, wherein the subject is afflictedwith a metabolic disorder.

35. The method of aspect 34, wherein the subject is afflicted with oneor more of the following: obesity, type 1 and type 2 diabetes mellitus,pancreatitis, dyslipidemia, nonalcoholic steatohepatitis (NASH), insulinresistance, hyperinsulinemia, glucose intolerance, hyperglycemia, andmetabolic syndrome.

36. A nucleic acid coding for one or more of the antibodies according toany one of the previous aspects, or for a VL and/or VH of any one of theantibodies.

37. A nucleic acid comprising a sequence with at least 90% identity tothe sequences set forth in Table 1.

38. A nucleic acid comprising a sequence with at least 95% identity tothe sequences set forth in Table 1.

39. A nucleic acid comprising a sequence set forth in Table 1.

40. A vector comprising the nucleic acid according to aspect 36, 37, 38,or 39.

41. A host cell comprising the vector of aspect 40.

42. A pharmaceutical composition comprising an antibody orantigen-binding fragment according to any one of aspects 1-23 for use intreating a metabolic disorder.

43. A method of making an antibody or antigen-binding fragment thereofwhich binds β-klotho, comprising the step of culturing the host cell ofaspect 41 under conditions suitable for expression of the antibody or afragment thereof

44. The pharmaceutical composition of aspect 42, wherein the metabolicdisorder is obesity, diabetes, hypertriglyceridemia, or dyslipidemia.

45. A pharmaceutical composition comprising an antibody orantigen-binding fragment according to any one of aspects 1-23 for use intreating a cardiovascular disorder.

46. A pharmaceutical composition comprising an antibody orantigen-binding fragment according to any one of aspects 1-23 for use ina method of reducing body weight, a method of reducing appetite or foodintake, a method of reducing plasma triglyceride (TG) concentrations orplasma total cholesterol (TC) concentrations, in a subject.

47. Use of the antibody or antigen-binding fragment thereof according toany one of aspects 1-23 in the preparation of a medicament for treatinga metabolic disorder, for treating a cardiovascular disorder, forreducing body weight, for reducing appetite or food intake, for reducingplasma TG concentrations or plasma TC concentrations, in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : pERK activation of human (FIG. 1A) and cynomolgus monkey (FIG.1B) FGFR1c_β-klotho_HEK293 cells by NOV004, NOV005, and NOV006. The pERKactivation data indicates that (i) NOV004 activated the human andcynomolgus monkey FGFR1 c_β-klotho receptor complex with EC₅₀ of about 3nM and 20 nM, respectively; (ii) NOV005 activated the human andcynomolgus monkey FGFR1c_β-klotho receptor complex with EC₅₀ of about 3nM and 16 nM, respectively; and (iii) NOV006 activated the human andcynomolgus monkey FGFR1c_β-klotho receptor complex with EC₅₀ of about 4nM and 18 nM, respectively.

FIGS. 2A-2C: Profiling of NOV004, NOV005, and NOV006 for pERK activationof human FGFR2c_β-klotho (FIG. 2A), FGFR3c_β-klotho (FIG. 2B), andFGFR4_β-klotho (FIG. 2C) HEK293 cells. FGF21 was used as a positivecontrol for activation of FGFR2c_β-klotho or FGFR3c_β-klotho. FGF19 wasused as a positive control for activation of FGFR4 β-klotho.

FIG. 3 : Profiling of NOV004, NOV005, and NOV006 for FGF23 activityusing HEK293 cells transfected with α-klotho, Egrl-luciferase andRenilla luciferase. FGF23 was used as positive control.

FIG. 4 : Competitive binding activity of NOV005 and NOV006 againstNOV004 for human β-klotho. Forte Bio® Biosensor system was used todetermine competitive binding activity to human β-klotho. In step 1,recombinant human β-klotho was loaded onto the senor, followed byloading of NOV004 until saturation in step 2. Then NOV005 or NOV006 wereloaded and competitive binding activity against NOV004 were detected.The absence of a second binding signal indicates that the antibodiescompete for binding to human β-klotho. An unrelated antibody was used asa negative control, and NOV004 was used as a positive control forself-competition.

FIGS. 5A-5C: NOV004, NOV005, and NOV006 concentration-time profilesfollowing IV injection in rats. Animals exhibited mean C_(max) ofapproximately 200 μg/mL at 1 h after IV administration of NOV004 (FIG.5A), NOV005 (FIG. 5B), or NOV006 (FIG. 5C), with all three antibodiesshowing comparable PK profiles.

FIG. 6A: Two subcutaneous 1 mg/kg doses of NOV005 (n=5 animals) orvehicle (n=3 animals) were administrated to male normoglycemic obesecynomolgus monkeys one week apart (days of dosing indicated by arrows).Food consumption data for standard chow was normalized as a percent ofbaseline with group mean±SEM. Monkeys consumed fruit, vegetables andpeanuts as treats throughout the study.

FIG. 6B: Two subcutaneous 1 mg/kg doses of NOV005 (n=5 animals) orvehicle (n=3 animals) were administrated to male normoglycemic obesecynomolgus monkeys one week apart (days of dosing indicated by arrows).Baseline body weights for NOV005 and vehicle treated animals were11.3+1.2 and 11.4+1.3 kg, respectively. Body weight data was normalizedas a percent of baseline with (A) group mean±SEM and (B) individualanimal data shown.

DETAILED DESCRIPTION

The present disclosure is based, in part, on the discovery of antibodymolecules that specifically bind to β-klotho and lead to activation ofFGF receptors, e.g., FGFR1c, and the activation of FGF21-mediatedsignaling (e.g., FGF21-receptor-dependent signaling). The presentdisclosure relates to both full IgG format antibodies as well asantigen-binding fragments thereof, such as Fab fragments (e.g.,antibodies NOV005 or NOV006).

Accordingly, the present disclosure provides antibodies thatspecifically bind to β-klotho (e.g., human and cynomolgus monkey(β-klotho), pharmaceutical compositions, production methods, and methodsof use of such antibodies and compositions.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this present disclosure pertains.

As used herein, the term “FGF21” refers to a member of the fibroblastgrowth factor (FGF) protein family. An exemplary amino acid sequence ofFGF21 (GenBank Accession No. NP_061986.1) is set forth as SEQ ID NO:1,the corresponding polynucleotide sequence of which is set forth as SEQID NO:2 (NCBI reference sequence number NM_019113.2).

As used herein, the term “FGF21 receptor” refers to a receptor for FGF21(Kharitonenkov, A, et al. (2008) Journal of Cellular Physiology 215:1-7;Kurosu, H, et al. (2007) JBC 282:26687-26695; Ogawa, Y, et al. (2007)PNAS 104:7432-7437).

The term “FGF21 polypeptide” refers to a naturally-occurring polypeptideexpressed in humans. For purposes of this disclosure, the term “FGF21polypeptide” can be used interchangeably to refer to any full-lengthFGF21 polypeptide, e.g., SEQ ID NO: 1, which consists of 209 amino acidresidues and which is encoded by the nucleotide sequence of SEQ ID NO:2;any mature form of the polypeptide, which consists of 181 amino acidresidues, and in which the 28 amino acid residues at the amino-terminalend of the full-length FGF21 polypeptide (i.e., which constitute thesignal peptide) have been removed, and variants thereof.

The term “antibody” as used herein means a whole antibody and anyantigen-binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. A whole antibody is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding portion” or “antigen-binding fragment” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., β-klotho). Antigen-binding functions of an antibody can beperformed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term antigen-binding portion orantigen-binding fragment of an antibody include a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; aF(ab)₂ fragment, a bivalent fragment comprising two Fab fragments linkedby a disulfide bridge at the hinge region; an Fd fragment consisting ofthe VH and CH1 domains; an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody; a single domain antibody (dAb)fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VHdomain or a VL domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by an artificial peptide linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl.Acad. Sci. 85:5879-5883). Such single chain antibodies include one ormore antigen-binding portions or fragments of an antibody. Theseantibody fragments are obtained using conventional techniques known tothose of skill in the art, and the fragments are screened for utility inthe same manner as are intact antibodies.

Antigen-binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005,Nature Biotechnology, 23, 9, 1126-1136). Antigen-binding portions ofantibodies can be grafted into scaffolds based on polypeptides such asFibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen-binding fragments can be incorporated into single chainmolecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen-binding regions (Zapata et al. (1995) Protein Eng.8(10):1057-1062; and U.S. Pat. No. 5,641,870).

As used herein, the term “affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity. As used herein,the term “high affinity” for an antibody or antigen-binding fragmentsthereof (e.g., a Fab fragment) generally refers to an antibody, orantigen-binding fragment, having a K_(D) of 10-9M or less.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an alpha carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “binding specificity” as used herein refers to the ability ofan individual antibody combining site to react with only one antigenicdeterminant.

The phrase “specifically (or selectively) binds” to an antibody (e.g., aβ-klotho-binding antibody) refers to a binding reaction that isdeterminative of the presence of a cognate antigen (e.g., a humanβ-klotho or cynomolgus β-klotho) in a heterogeneous population ofproteins and other biologics. The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

The term “FGF21 mediated” or similar refers to the fact that the FGF21receptor and/or the antibodies of the present disclosure mediate thecellular response and the FGF21 signaling pathway upon binding toβ-klotho, thereby triggering a variety of physiological effects,including but not limited to a reduction in one or more of thefollowing: plasma triglycerides, plasma insulin, plasma glucose, foodintake, and body weight.

An “FGF21-associated disorder,” “FGF21-associated condition,” “diseaseor condition associated with FGF21,” or similar terms as used herein,refer to any number of conditions or diseases for which the prevention,diagnosis, and/or treatment by activation of the FGF21 signaling pathway(e.g., by activation of FGF21 receptor signaling), is sought. These caninclude conditions, diseases, or disorders characterized by aberrantFGF21 signaling (e.g., aberrant activation of FGF21-mediated signalingand/or FGF21 receptor signaling). These conditions include but are notlimited to metabolic, endocrine, and cardiovascular disorders, such asobesity, type 1 and type 2 diabetes mellitus, pancreatitis,dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, metabolic syndrome, acute myocardialinfarction, hypertension, cardiovascular disease, atherosclerosis,peripheral arterial disease, stroke, heart failure, coronary heartdisease, kidney disease, diabetic complications, neuropathy,gastroparesis, disorders associated with severe inactivating mutationsin the insulin receptor, and other metabolic disorders, and in reducingthe mortality and morbidity of critically ill patients.

“Type 2 diabetes mellitus” is a condition characterized by excessglucose production in spite of the availability of insulin, andcirculating glucose levels remain excessively high as a result ofinadequate glucose clearance.

“Type 1 diabetes mellitus” is a condition characterized by high bloodglucose levels caused by total lack of insulin. This occurs when thebody's immune system attacks the insulin-producing beta cells in thepancreas and destroys them. The pancreas then produces little or noinsulin.

“Pancreatitis” is inflammation of the pancreas.

“Dyslipidemia” is a disorder of lipoprotein metabolism, includinglipoprotein overproduction or deficiency. Dyslipidemias may bemanifested by elevation of the total cholesterol, low-densitylipoprotein (LDL) cholesterol and triglyceride concentrations, and adecrease in high-density lipoprotein (HDL) cholesterol concentration inthe blood.

“Nonalcoholic steatohepatitis (NASH)” is a liver disease, not associatedwith alcohol consumption, characterized by fatty change of hepatocytes,accompanied by intralobular inflammation and fibrosis.

“Glucose intolerance,” or Impaired Glucose Tolerance (IGT) is apre-diabetic state of dysglycemia that is associated with increased riskof cardiovascular pathology. The pre-diabetic condition prevents asubject from moving glucose into cells efficiently and utilizing it asan efficient fuel source, leading to elevated glucose levels in bloodand some degree of insulin resistance.

“Hyperglycemia” is defined as an excess of sugar (glucose) in the blood.

“Hypoglycemia”, also called low blood sugar, occurs when your bloodglucose level drops too low to provide enough energy for your body'sactivities.

“Hyperinsulinemia” is defined as a higher-than-normal level of insulinin the blood.

“Insulin resistance” is defined as a state in which a normal amount ofinsulin produces a subnormal biologic response.

“Obesity,” in terms of the human subject, can be defined as that bodyweight over 20 percent above the ideal body weight for a givenpopulation (R. H. Williams, Textbook of Endocrinology, 1974, p.904-916). It can also be defined as a Body Mass Index (BMI, defined as aperson's weight in kilograms divided by the square of his height inmeters (kg/m2)) as greater than or equal to 30.

“Metabolic syndrome” can be defined as a cluster of at least three ofthe following signs: abdominal fat—in most men, a 40-inch waist orgreater; high blood sugar—at least 110 milligrams per deciliter (mg/dl)after fasting; high triglycerides—at least 150 mg/dL in the bloodstream;low HDL—less than 40 mg/dl; and, blood pressure of 130/85 mmHg orhigher.

“Hypertension” or high blood pressure that is a transitory or sustainedelevation of systemic arterial blood pressure to a level likely toinduce cardiovascular damage or other adverse consequences.

Hypertension has been arbitrarily defined as a systolic blood pressureabove 140 mmHg or a diastolic blood pressure above 90 mmHg.

“Cardiovascular diseases” are diseases related to the heart or bloodvessels.

“Peripheral arterial disease” occurs when plaque builds up in thearteries that carry blood to the head, organs and limbs. Over time,plaque can harden and narrow the arteries which limits the flow ofoxygen-rich blood to organs and other parts of the body.

“Atherosclerosis” is a vascular disease characterized by irregularlydistributed lipid deposits in the intima of large and medium-sizedarteries, causing narrowing of arterial lumens and proceeding eventuallyto fibrosis and calcification. Lesions are usually focal and progressslowly and intermittently. Limitation of blood flow accounts for mostclinical manifestations, which vary with the distribution and severityof lesions.

“Stroke” is any acute clinical event, related to impairment of cerebralcirculation, that lasts longer than 24 hours. A stroke involvesirreversible brain damage, the type and severity of symptoms dependingon the location and extent of brain tissue whose circulation has beencompromised.

“Heart failure”, also called congestive heart failure, is a condition inwhich the heart can no longer pump enough blood to the rest of the body.

“Coronary heart disease”, also called coronary artery disease, is anarrowing of the small blood vessels that supply blood and oxygen to theheart.

“Kidney disease” or nephropathy is any disease of the kidney. Diabeticnephropathy is a major cause of morbidity and mortality in people withtype 1 or type 2 diabetes mellitus.

“Diabetic complications” are problems, caused by high blood glucoselevels, with other body functions such as kidneys, nerves(neuropathies), feet (foot ulcers and poor circulation) and eyes (e.g.retinopathies). Diabetes also increases the risk for heart disease andbone and joint disorders. Other long-term complications of diabetesinclude skin problems, digestive problems, sexual dysfuntion andproblems with teeth and gums.

“Neuroapathies” are any diseases involving the cranial nerves or theperipheral or autonomic nervous system.

“Gastroparesis” is weakness of gastric peristalsis, which results indelayed emptying of the bowels.

The critically ill patients encompassed by the present disclosuregenerally experience an unstable hypermetabolic state. This unstablemetabolic state is due to changes in substrate metabolism, which maylead to relative deficiencies in some nutrients. Generally there is anincreased oxidation of both fat and muscle.

Moreover, critically ill patients are preferably patients thatexperience systemic inflammatory response syndrome or respiratorydistress. A reduction in morbidity means reducing the likelihood that acritically ill patient will develop additional illnesses, conditions, orsymptoms or reducing the severity of additional illnesses, conditions,or symptoms. For example reducing morbidity may correspond to a decreasein the incidence of bacteremia or sepsis or complications associatedwith multiple organ failure.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of thepresent disclosure. The following eight groups contain amino acids thatare conservative substitutions for one another: 1) Alanine (A), Glycine(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). In someembodiments, the term “conservative sequence modifications” are used torefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies of thepresent disclosure may include amino acid residues not encoded by humansequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo).

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

A “humanized” antibody is an antibody that retains the reactivity of anon-human antibody while being less immunogenic in humans. This can beachieved, for instance, by retaining the non-human CDR regions andreplacing the remaining parts of the antibody with their humancounterparts (i.e., the constant region as well as the frameworkportions of the variable region). See, e.g., Morrison et al., Proc.Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv.Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536,1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec.Immun., 31:169-217, 1994. Other examples of human engineering technologyinclude, but are not limited to Xoma technology disclosed in U.S. Pat.No. 5,766,886.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,WI), or by manual alignment and visual inspection (see, e.g., Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(Ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableon the world wide web at gcg.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities (e.g.,an isolated antibody that specifically binds β-klotho is substantiallyfree of antibodies that specifically bind antigens other than β-klotho).An isolated antibody that specifically binds β-klotho may, however, havecross-reactivity to other antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgGsuch as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Isotype also includes modified versions of one of theseclasses, where modifications have been made to alter the Fc function,for example, to enhance or reduce effector functions or binding to Fcreceptors.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term k_(dis)” or “k_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “I(D”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of k_(d) tok_(a) (i.e. k_(d)/k_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. Methods for determining the K_(D) of an antibodyinclude measuring surface plasmon resonance using a biosensor systemsuch as a Biacore® system, or measuring affinity in solution by solutionequilibrium titration (SET).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98,1994).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, the termrefers to the functional relationship of a transcriptional regulatorysequence to a transcribed sequence. For example, a promoter or enhancersequence is operably linked to a coding sequence if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO)or a human cell. The optimized nucleotide sequence is engineered toretain completely or as much as possible the amino acid sequenceoriginally encoded by the starting nucleotide sequence, which is alsoknown as the “parental” sequence. The optimized sequences herein havebeen engineered to have codons that are preferred in mammalian cells.However, optimized expression of these sequences in other eukaryoticcells or prokaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates (e.g.: mammals and non-mammals) such as,non-human primates (e.g.: cynomolgus monkey), sheep, dog, cow, chickens,amphibians, and reptiles. Except when noted, the terms “patient” or“subject” are used herein interchangeably. As used herein, the terms“cyno” or “cynomolgus” refer to the cynomolgus monkey (Macacafascicularis).

As used herein, the term “treating” or “treatment” of any disease ordisorder (e.g., FGF21 associated disorder) refers in one embodiment, toameliorating the disease or disorder (i.e., slowing or arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treating” or “treatment” refers to modulating thedisease or disorder, either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. In yet another embodiment, “treating” or“treatment” refers to preventing or delaying the onset or development orprogression of the disease or disorder.

“Prevention” as it relates to indications described herein, including,e.g., FGF21 associated disorder, means any action that prevents or slowsa worsening in e.g., FGF21 associated disease parameters, as describedbelow, in a patient at risk for said worsening.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, such as anadeno-associated viral vector (AAV, or AAV2), wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”).

In general, expression vectors of utility in recombinant DNA techniquesare often in the form of plasmids. In the present specification,“plasmid” and “vector” may be used interchangeably as the plasmid is themost commonly used form of vector. However, the present disclosure isintended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

“Modulation of FGF21 activity,” as used herein, refers to an increase ordecrease in FGF21 activity that can be a result of, for example,interaction of an agent with an FGF21 polynucleotide or polypeptide,activation of the FGF21 signaling pathway and/or activation ofFGF21-mediated signaling (e.g., FGF21-receptor-dependent signaling), andthe like. For example, modulation of a biological activity refers to anincrease or a decrease in a biological activity. FGF21 activity can beassessed by means including, without limitation, assaying blood glucose,insulin, triglyceride, or cholesterol levels in a subject; by assessingpolypeptide levels of beta-klotho and/or FGF receptors (e.g., FGFR-1c);or by assessing activation of FGF21-mediated signaling (e.g., ofFGF21-receptor-dependent signaling).

Comparisons of FGF21 activity can also be accomplished by, e.g.,measuring levels of an FGF21 downstream biomarker, and measuringincreases in FGF21 signaling. Activity can also be assessed bymeasuring: cell signaling; kinase activity; glucose uptake intoadipocytes; blood insulin, triglyceride, or cholesterol levelfluctuations; liver lipid or liver triglyceride level changes;interactions between FGF21 and/or beta-klotho and an FGF21 receptor; orphosphorylation of an FGF21 receptor. In some embodimentsphosphorylation of an FGF21 receptor can be tyrosine phosphorylation. Insome embodiments modulation of FGF21 activity can cause modulation of anFGF21-related phenotype.

An “FGF21 downstream biomarker,” as used herein, is a gene or geneproduct, or measurable indicia of a gene or gene product. In someembodiments, a gene or activity that is a downstream marker of FGF21exhibits an altered level of expression, or in a vascular tissue. Insome embodiments, an activity of the downstream marker is altered in thepresence of an FGF21 modulator. In some embodiments, the downstreammarkers exhibit altered levels of expression when FGF21 is perturbedwith an FGF21 modulator of the present disclosure. FGF21 downstreammarkers include, without limitation, glucose or 2-deoxy-glucose uptake,pERK and other phosphorylated or acetylated proteins or NAD levels.

As used herein, the term “up-regulates” refers to an increase,activation or stimulation of an activity or quantity. For example, inthe context of the present disclosure, FGF21 modulators may increase theactivity of beta-klotho and/or an FGF21 receptor. In one embodiment,FGFR-1c may be upregulated in response to an FGF21 modulator.Upregulation can also refer to an FGF21-related activity, such as e.g.,the ability to lower blood glucose, insulin, triglyceride, orcholesterol levels; to reduce liver lipid or triglyceride levels; toreduce body weight; to improve glucose tolerance, energy expenditure, orinsulin sensitivity; or to cause phosphorylation of an FGF21 receptor;or to increase an FGF21 downstream marker. The FGF21 receptor can beβ-klotho and FGFR-1c. Up-regulation may be at least 25%, at least 50%,at least 75%, at least 100%, at least 150%, at least 200%, at least250%, at least 400%, or at least 500% as compared to a control.

As used herein, the term “modulator” refers to a composition thatmodulates one or more physiological or biochemical events associatedwith an FGF21-associated disorder, such as type 1 or type 2 diabetesmellitus or a metabolic condition like obesity. Said events include butare not limited to the ability to lower blood glucose, insulin,triglyceride, or cholesterol levels; to reduce liver lipid or livertriglyceride levels; to reduce body weight; and to improve glucosetolerance, energy expenditure, or insulin sensitivity.

FGF21 Proteins

The present disclosure provides FGF21 mimetic antibodies (e.g.,monoclonal antibodies that bind to beta-klotho (β-klotho)) that caninduce FGF21-mediated signaling (e.g., FGF21-receptor-mediatedsignaling), as defined herein. in vivo, the mature form of FGF21 is theactive form of the molecule. An exemplary human FGF21 wild-type sequencehas NCBI reference sequence number NP_061986.1, and can be found in suchissued patents as, e.g., U.S. Pat. No. 6,716,626 B1 for example, as setforth below (SEQ ID NO 1).

Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1               5                  10                  15Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro            20                  25                  30Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr        35                  40                  45Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg    50                  55                  60Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu65                  70                  75                  80Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val                 85                  90                  95Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly            100                 105                 110Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu        115                 120                 125Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu    130                 135                 140His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly145                 150                 155                 160Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu                165                 170                 175Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp            180                 185                 190Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala        195                 200                 205 Ser 209

The corresponding mRNA sequence coding for the full-length FGF21polypeptide (NCBI reference sequence number NM_019113.2) is shown below(SEQ ID NO. 2)

1 ctgtcagctg aggatccagc cgaaagagga gccaggcact caggccacct gagtctactc 61acctggacaa ctggaatctg gcaccaattc taaaccactc agcttctccg agctcacacc 121ccggagatca cctgaggacc cgagccattg atggactcgg acgagaccgg gttcgagcac 181tcaggactgt gggtttctgt gctggctggt cttctgctgg gagcctgcca ggcacacccc 241atccctgact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctccac 301acagatgatg cccagcagac agaagcccac ctggagatca gggaggatgg gacggtgggg 361ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 421attcaaatct tgggagtcaa gacatccagg ttcctgtgcc agcggccaga tggggccctg 481tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 541ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 601tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact accaggcctg 661ccccccgcac tcccggagcc acccggaatc ctggcccccc agccccccga tgtgggctcc 721tcggaccctc tgagcatggt gggaccttcc cagggccgaa gccccagcta cgcttcctga 781agccagaggc tgtttactat gacatctcct ctttatttat taggttattt atcttattta 841tttttttatt tttcttactt gagataataa agagttccag aggagaaaaa aaaaaaaaaa 901aaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

The mature FGF21 sequence lacks a leader sequence and may also includeother modifications of a polypeptide such as proteolytic processing ofthe amino terminus (with or without a leader sequence) and/or thecarboxyl terminus, cleavage of a smaller polypeptide from a largerprecursor, N-linked and/or O-linked glycosylation, and otherpost-translational modifications understood by those with skill in theart. A representative example of a mature FGF21 sequence has thefollowing sequence (SEQ ID NO: 53, which represents amino acid positions29-209 of full length FGF21 protein sequence (NCBI reference sequencenumber NP_061986.1)):

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val                 5                  10                  15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His            20                  25                  30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser        35                  40                  45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln    50                  55                  60Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly65                  70                  75                  80Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg                85                  90                  95Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His            100                 105                 110Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro        115                 120                 125Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro    130                 135                 140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val145                 150                 155                 160Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser                165                 170                 175Pro Ser Tyr Ala Ser

The corresponding cDNA sequence coding for a mature FGF21 polypeptide(SEQ ID NO:53) is shown below (SEQ ID NO: 63):

1 caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 61ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 121gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 181ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 301gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 421ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 481ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 541tcctga

FGF21 Mimetic Antibodies & Antigen-Binding Fragments

The present disclosure provides antibodies that specifically bind toβ-klotho (e.g., human β-klotho). In some embodiments, the presentdisclosure provides antibodies that specifically bind to human andcynomolgus monkey β-klotho. Antibodies of the present disclosureinclude, but are not limited to, the human monoclonal antibodies andFabs, isolated as described in the Examples.

The β-klotho wild-type sequence has NCBI reference sequence numberNP_783864.1, and can be found in such literature as Xu, et al. (2007) JBiol Chem. 282(40):29069-72 and Lin, et al. (2007) J Biol Chem.282(37):27277-84. The full-length cDNA encoding human β-klotho hasGenBank Accession number NM_175737). The protein sequence is as follows(SEQ ID NO:52).

1 mkpgcaagsp gnewiffstd eirtryrntm sngglqvrsvi lsalillrav tgfagdgrai 61wsknpnftpv nesqlflydt fpk

ffwgig tgalqvegsw kkdgkgpsiw dhfihthlkn 121vsstngssds yiflekdlsa ldfigvsfyq fsiswprlfp dgivtvanak glqyystlld 181alvlrniepi vtlyhwdlpl alqekyggwk ndtiidifnd yatycfqmfg drvkywitih 241npylvawhqy gtgmhapgek gnlaavytvg hnlikahskv whnynthfrp hqkgwlsitl 301gshwiepnrs ent

difkcq gsmvsvlgwf anpihgdgdy pegmrkklfs vlp

fseaek 361 hemrgtadff afsfgpnnfk plntmakmgq nvslnlreal

wikleynnp rillaengwf 421 tdsrvktedt talymmknfl sqvlqairld eirvtgytawslldgfewqd aytirrglfy 481 ydfnskqker kpkssahyyk qiirengfsl kestpdvqqqfpcdfswgvt eavlkpesva 541

pqfsdphl yvwnatgnrl lhrvegvrlk trpaqctdfv nikkqlemla rmkvthyrfa 601 ldw

vlptg nlsavnrqal ryyrcvv

eg lklgisemvt lyypthahlg lpepllhadg 661 wlnpst

eaf qayaglcfqe lgdlvklwit inepnrladi ynrsgndtyg aahnllvaha 721lawrlydrqf rpsqrgavsl slhadwaepa npyadshwra aerflqfeia wfaeplfktg 781dypaamreyi askhrrglss salprlteae rrllkgtvdf calnhfttrf vmheqlagsr 841ydsdrdiqfl qditrlsspt rlavipwgvr kllrwvrrny gdmdiyitas giddqaledd 901rlrkyylgky lqevlkayli dkvridgyya fklaeekakp rfgfftsdfk akssiqfynk 961vissrgfp

e nsssrcsqtq entectvclf lvqkkplifl gccffstlvl llsialfqrq 1021krrkfwkakn lqhiplkkgk rvvs

indicates data missing or illegible when filed

The present disclosure provides antibodies and antigen-binding fragmentsthereof that specifically bind a β-klotho protein (e.g., human and/orcynomolgus monkey β-klotho) and is capable of activating or increasingthe activity of a β-klotho/FGFR1c receptor complex, wherein the antibodyis described in Table 1, e.g., NOV005 or NOV006. In specific aspects,antibodies and antigen-binding fragments thereof provided herein, thatspecifically bind a β-klotho protein (e.g., human and/or cynomolgusmonkey (β-klotho) and is capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex, is not an antibodydescribed in Table 2, for example, NOV001, NOV002, NOV003, or NOV004.Antibodies described in Table 2 have been described in PCT InternationalPatent Application No. PCT/IB2016/054660 filed on Aug. 2, 2016, which isincorporated by reference herein in its entirety.

The present disclosure provides antibodies that specifically bind aβ-klotho protein (e.g., human and/or cynomolgus monkey β-klotho),wherein the antibodies comprise a VH domain having an amino acidsequence of SEQ ID NO: 15. The present disclosure also providesantibodies that specifically bind to a β-klotho protein, wherein theantibodies comprise a VH CDR having an amino acid sequence of any one ofthe VH CDRs listed in Table 1, infra. In particular, the presentdisclosure provides antibodies that specifically bind to a β-klothoprotein (e.g., human and cynomolgus monkey β-klotho), wherein theantibodies comprise (or alternatively, consist of) one, two, three, ormore VH CDRs having an amino acid sequence of any of the VH CDRs listedin Table 1, infra.

The present disclosure provides antibodies that specifically bind to aβ-klotho protein, said antibodies comprising a VL domain having an aminoacid sequence of SEQ ID NOs: 26 or 32. The present disclosure alsoprovides antibodies that specifically bind to a β-klotho protein (e.g.,human and cynomolgus monkey β-klotho), said antibodies comprising a VLCDR having an amino acid sequence of any one of the VL CDRs listed inTable 1, infra. In particular, the present disclosure providesantibodies that specifically bind to a β-klotho protein (e.g., human andcynomolgus monkey β-klotho), said antibodies comprising (oralternatively, consisting of) one, two, three or more VL CDRs having anamino acid sequence of any of the VL CDRs listed in Table 1, infra.

Other antibodies of the present disclosure include amino acids that havebeen mutated, yet have at least 60, 70, 80, 85, 90 or 95 percentidentity in the CDR regions with the CDR regions depicted in thesequences described in Table 1. In some embodiments, it includes mutantamino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acidshave been mutated in the CDR regions when compared with the CDR regionsdepicted in the sequence described in Table 1.

The present disclosure also provides nucleic acid molecules orpolynucleotides comprising nucleic acid sequences that encode VH, VL,the full length heavy chain, and the full length light chain of theantibodies that specifically bind to a β-klotho protein (e.g., human andcynomolgus monkey β-klotho). Such nucleic acid sequences can beoptimized for expression in mammalian cells (for example, Table 1 showsthe optimized nucleic acid sequences for the heavy chain and light chainof antibodies of the present disclosure).

TABLE 1 Examples of FGF21 MiMetic Antibodies and Fabs SEQ ID NO:Amino acid sequence NOV005 HCDR1 (Combined) 6 GYSITSGYTWHHCDR2 (Combined) 7 YIHYSVYTNYNPSLKS HCDR3 (Combined) 8 RTTSLERYFDVHCDR1 (Kabat) 9 SGYTWH HCDR2 (Kabat) 7 YIHYSVYTNYNPSLKS HCDR3 (Kabat) 8RTTSLERYFDV HCDR1 (Chothia) 10 GYSITSGY HCDR2 (Chothia) 11 HYSVYHCDR3 (Chothia) 8 RTTSLERYFDV HCDR1 (IMGT) 12 GYSITSGYT HCDR2 (IMGT) 13IHYSVYT HCDR3 (IMGT) 14 ARRTTSLERYFDV VH 15QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWIGYIHYSVYTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARRTTSLERYFDVWGQGTLVTVSS DNA VH (vector 1) 16CAAGTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCCTAGCCAGACCCTGTCCCTGACCTGCACCGTGTCCGGCTACTCCATCACCTCCGGCTACACCTGGCACTGGATCCGGCAGCACCGGGGCAAGGGCCTGGAATGGATCGGCTACATCCACTACTCCGTGTACACCAACTACAACCCCAGCCTGAAGTCCAGAGTGACCATCTCCCGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACCGCCGCTGACACCGCCGTGTACTACTGCGCCAGACGGACCACCTCCCTGGAACGGTACTTCGACGTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT DNA VH (vector 2) 36CAAGTCCAGCTGCAAGAATCCGGACCCGGCCTCGTCAAGCCGTCCCAGACTCTGTCTCTCACTTGCACGGTGTCAGGCTACAGCATCACCAGCGGTTACACCTGGCACTGGATCAGGCAGCATCCTGGAAAGGGGCTGGAATGGATTGGGTACATTCACTACTCGGTGTACACCAACTACAACCCATCGCTCAAGTCGAGAGTCACCATTTCCCGGGACACCTCCAAGAACCAGTTCAGCCTCAAGCTGTCCTCTGTGACCGCCGCTGATACTGCCGTGTACTATTGCGCACGCCGGACTACTTCCCTGGAGCGCTACTTCGACGTCTGGGGCCAGGGCACTTTGGTCACCGTCAGCTCC Heavy Chain 17QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWIGYIHYSVYTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARRTTSLERYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DNA Heavy Chain 18CAAGTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCCTAGC (vector 1)CAGACCCTGTCCCTGACCTGCACCGTGTCCGGCTACTCCATCACCTCCGGCTACACCTGGCACTGGATCCGGCAGCACCCCGGCAAGGGCCTGGAATGGATCGGCTACATCCACTACTCCGTGTACACCAACTACAACCCCAGCCTGAAGTCCAGAGTGACCATCTCCCGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACCGCCGCTGACACCGCCGTGTACTACTGCGCCAGACGGACCACCTCCCTGGAACGGTACTTCGACGTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCTTCCACCAAGGGGCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGGGCGCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAGGGGGTGGAAGCCAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCCCCTGAGCTGCTGGGGGGACCTTCCGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTGTCCAACAAGGCCCTGGCCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACACTGCCTCCCAGCCGGGAAGAGATGACCAAGAATCAAGTGTCCCTGACATGTCTGGTCAAGGGCTTCTACCCTAGCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCCCCTGGCAAG DNA Heavy Chain 37CAAGTCCAGCTGCAAGAATCCGGACCCGGCCTCGTCAAGCCGTCC (vector 2)CAGACTCTGTCTCTCACTTGCACGGIGTCAGGCTACAGCATCACCAGCGGTTACACCTGGCACTGGATCAGGCAGCATCCTGGAAAGGGGCTGGAATGGATTGGGTACATTCACTACTCGGTGTACACCAACTACAACCCATCGCTCAAGTCGAGAGTCACCATTTCCCGGGACACCTCCAAGAACCAGTTCAGCCTCAAGCTGTCCTCTGTGACCGCCGCTGATACTGCCGTGTACTATTGCGCACGCCGGACTACTTCCCTGGAGCGCTACTTCGACGTCTGGGGCCAGGGCACTTTGGTCACCGTCAGCTCCGCCAGCACTAAGGGCCCCAGCGTGTTTCCGCTGGCCCCCTCCTCCAAAAGCACCTCCGGCGGAACTGCCGCGCTCGGATGTCTCGTGAAGGACTATTTCCCCGAGCCTGTGACAGTGTCATGGAACTCGGGAGCACTGACCAGCGGAGTGCATACTTTTCCCGCGGTCCTGCAGTCCTCCGGATTGTACAGCCTGTCATCGGTCGTGACCGTGCCGTCCTCATCGCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAACCTAGCAACACCAAAGTGGATAAGCGGGTGGAACCTAAGTCCTGCGACAAGACTCACACTTGTCCGCCATGCCCAGCGCCTGAACTCCTGGGTGGTCCTTCGGTGTTCCTGTTCCCGCCAAAGCCGAAGGACACCCTGATGATCTCCCGGACGCCTGAAGTGACCTGTGTGGTGGTGGCTGTGTCACATGAGGACCCTGAAGTCAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGCGTCGTGTCGGTGCTGACCGTGTTGCACCAAGACTGGCTGAATGGAAAGGAGTATAAGTGCAAAGTGTCCAACAAGGCCCTGGCCGCACCAATTGAGAAAACCATCTCCAAGGCCAAGGGACAGCCGCGCGAACCCCAAGTGTACACCCTTCCCCCGTCCCGGGAGGAAATGACCAAGAATCAAGTCTCCCTGACTTGCCTTGTGAAGGGTTTCTACCCCTCCGAGATCGCCGTGGAGTGGGAGTCAAACGGGCAGCCGGAAAACAACTACAAGACCACACCTCCGGTGCTGGATTCCGACGGCTCCTTCTTCTTGTACTCGAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCACAACCACTACACTCAGAAGTCGCTCTCGCTGAGCCCCGGGAAG LCDR1 (Combined) 19QASQDISNYLN LCDR2 (Combined) 20 YTSRLQS LCDR3 (Combined) 21 QQGNTLPYTLCDR1 (Kabat) 19 QASQDISNYLN LCDR2 (Kabat) 20 YTSRLQS LCDR3 (Kabat) 21QQGNTLPYT LCDR1 (Chothia) 22 SQDISNY LCDR2 (Chothia) 23 YTSLCDR3 (Chothia) 24 GNTLPY LCDR1 (IMGT) 25 QDISNY LCDR2 (IMGT) 23 YTSLCDR3 (IMGT) 21 QQGNTLPYT VL 26DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGADYTFTISSLQPEDIATYFCQQ GNTLPYTFGQGTKLEIKDNA VL (vector 1) 54 GACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCAGGCCTCCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCTCCCGGCTGCAGTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCTGGCGCCGACTACACCTTCACCATCTCCAGCCTGCAGCCCGAGGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAA ATCAAG DNA VL (vector 2)27 GACATCCAGATGACCCAGAGCCCGTCGTCCCTCTCCGCTTCCGTGGGAGATAGAGTGACCATCACCTGTCAAGCCAGCCAGGATATTTCAAACTACCTGAATTGGTACCAGCAGAAGCCGGGGAAGGCTCCCAAGTTGCTCATCTACTACACATCGAGGCTGCAGTCCGGCGTGCCCAGCCGGTTCTCCGGGTCCGGATCAGGCGCCGACTATACCTTCACCATTTCCTCCCTGCAACCGGAGGACATTGCCACTTACTTCTGCCAACAAGGGAACACCCTGCCCTACACTTTCGGACAAGGAACTAAGCTGGAA ATCAAG Light Chain 28DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DNA Light Chain 29GACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCTCCGTG (vector 1)GGCGACAGAGTGACCATCACCTGTCAGGCCTCCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCTCCCGGCTGCAGTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCTGGCGCCGACTACACCTTCACCATCTCCAGCCTGCAGCCCGAGGATATCGCTACCTACTTCTGTCACCAAGGCAACACCCTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCTCGCGAGGCCAAAGTGCAGTGGAAAGTGGACAAGGCCCTGCAGAGGGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCGTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAAC CGGGGCGAGTGCDNA Light Chain 51 GACATCCAGATGACCCAGAGCCCGTCGTCCCTCTCCGCTTCCGTG(vector 2) GGAGATAGAGTGACCATCACCTGTCAAGCCAGCCAGGATATTTCAAACTACCTGAATTGGTACCAGCAGAAGCCGGGGAAGGCTCCCAAGTTGCTCATCTACTACACATCGAGGCTGCAGTCCGGCGTGCCCAGCCGGTTCTCCGGGTCCGGATCAGGCGCCGACTATACCTTCACCATTTCCTCCCTGCAACCGGAGGACATTGCCACTTACTTCTGCCAACAAGGGAACACCCTGCCCTACACTTTCGGAGAAGGAACTAAGCTGGAAATCAAGCGTACGGTGGCCGCGCCGTTCGTGTTCATCTTCCCTCCTTCTGACGAGCAGCTCAAGAGCGGCACCGCGTCGGTGGTCTGCCTGCTGAACAACTTCTACCCACGGGAGGCCAAGGTCCAGTGGAAAGTGGATAACGCATTGCAGTCGGGAAACTCACAGGAGTCGGTGACCGAACAGGACTCCAAAGACTCAACCTACTCCCTGTCCTCCACTCTTACCCTGTCCAAGGCGGACTACGAAAAGCACAAGGTCTACGCCTGCGAAGTGACCCATCAGGGTCTGAGCAGCCCTGTGACTAAGAGCTTTAAC CGCGGCGAATGC NOV006HCDR1 (Combined) 6 GYSITSGYTWH HCDR2 (Combined) 7 YIHYSVYTNYMPSLKSHCDR3 (Combined) 8 RTTSLEPYFDV HCDR1 (Kabat) 9 SGYTWH HCDR2 (Kabat) 7YIHYSVYTNYNPSLKS HCDR3 (Kabat) 8 RTTSLERYFDV HCDR1 (Chothia) 10 GYSITSGYHCDR2 (Chothia) 11 HYSVY HCDR3 (Chothia) 8 RTTSLERYFDV HCDR1 (IMGT) 12GYSITSGYT HCDR2 (IMGT) 13 IHYSVIT HCDR3 (IMGT) 14 ARRTTSLERYFDV VH 15QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWIGYIHYSVYTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARRTTSLERYFDVWGQGTLVTVSS DNA VH (vector 1) 16CAAGTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCCTAGCCAGACCCTGTCCCTGACCTGCACCGTGTCCGGCTACTCCATCACCTCCGGCTACACCTGGCACTGGATCCGGCAGCACCCCGGCAAGGGCCTGGAATGGATCGGCTACATCCACTACTCCGTGTACACCAACTACAACCCCAGCCTGAAGTCCAGAGTGACCATCTCCCGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACCGCCGCTGACACCGCCGTGTACTACTGCGCCAGACGGACCACCTCCCTGGAACGGTACTTCGACGTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT DNA VH (vector 2) 38CAAGTCCAGCTGCAAGAATCCGGACCCGGCCTCGTCAAGCCGTCCCAGACTCTGTCTCTCACTTGCACGGTGTCAGGCTACAGCATCACCAGCGGTTACACCTGGCACTGGATCAGGCAGCATCCTGGAAAGGGGCTGGAATGGATTGGGTACATTCACTACTCGGTGTACACCAACTACAACCCATCGCTCAAGTCGAGAGTCACCATTTCCCGGGACACCTCCAAGAACCAGTTCAGCCTCAAGCTGTCCTCTGTGACCGCCGCTGATACTGCCGTGTACTATTGCGCACGCCGGACTACTTCCCTGGAGCGCTACTTCGACGTCTGGGGCCAGGGCACTTTGGTCACCGTCAGCTCC Heavy Chain 17QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWIGYIHYSVYTNYNPSLKSPVTISRDTSKNQFSLKLSSVTAADTAVYYCARRTTSLEPYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DNA Heavy Chain 30CAAGTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCCTAGC (vector 1)CAGACCCTGTCCCTGACCTGCACCGTGTCCGGCTACTCCATCACCTCCGGCTACACCTGGCACTGGATCCGGCAGCACCCCGGCAAGGGCCTGGAATGGATCGGCTACATCCACTACTCCGTGTACACCAACTACAACCCCAGCCTGAAGTCCAGAGIGACCATCTCCCGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACCGCCGCTGACACCGCCGTGTACTACTGCGCCAGACGGACCACCTCCCTGGAACGGTACTTCGACGTGTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCTTCCACCAAGGGGCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGGGGCACCGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCCCCTGAGCTGCTGGGGGGACCTTCCGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTGTCCAACAAGGCCCTGGCCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAAGTGTACACACTGCCTCCCAGCCGGGAAGAGATGACCAAGAATCAAGTGTCCCTGACATGTCTGGTCAAGGGCTTCTACCCTAGCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCCCCTGGCAAG DNA Heavy Chain 39CAAGTCCAGCTGCAAGAATCCGGACCCGGCCTCGTCAAGCCGTCC (vector 2)CAGACTCTGTCTCTCACTTGCACGGTGTCAGGCTACAGCATCACCAGCGGTTACACCTGGCACTGGATCAGGCAGCATCCTGGAAAGGGGCTGGAATGGATTGGGTACATTCACTACTCGGTGTACACCAACTACAACCCATCGCTCAAGTCGAGAGTCACCATTTCCCGGGACACCTCCAAGAACCAGTTCAGCCTCAAGCTGTCCTCTGTGACCGCCGCTGATACTGCCGTGTACTATTGGGCACGCCGGACTACTTCCCTGGAGCGCTACTTCGACCTCTGGGGCCAGGGCACTTTGGTCACCGTCAGCTCCGCCAGCACTAAGGGCCCCAGCGTGTTTCCGCTGGCCCCCTCCTCCAAAAGCACCTCCGGGGGAACTGCCGCGCTCGGATGTCTCGTGAAGGACTATTTCCCCGAGCCTGTGACAGTGTCATGGAACTCGGGAGCACTGACCAGCGGAGTGCATACTTTTCCCGCGGTCCTGCAGTCCTCCGGATTGTACAGCCTGTCATCGGTCGTGACCGTGCCGTCCTCATCGCTGGGCACCCAGACCTACATCTGCAACGIGAACCACAAACCTAGCAACACCAAAGTGGATAAGCGGGTGGAACCTAAGTCCTGCGACAAGACTCACACTTGTCCGCCATGCCCAGCGCCTGAACTCCTGGGTGGTCCTTCGGTGTTCCTGTTCCCGCCAAAGCCGAAGGACACCCTGATGATCTCCCGGACGCCTGAAGTGACCTGTGTGGTGGTGGCTGTGTCACATGAGGACCCTGAAGTCAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGCGTCGTGTCGGTGCTGACCGTGTTGCACCAAGACTGGCTGAATGGAAAGGAGTATAAGTGCAAAGTGTCCAAGAAGGCCCTGGCCGCACCAATTGAGAAAACCATCTCCAAGGCCAAGGGACAGCCGCGCGAACCCCAAGTGTACACCCTTCCCCCGTCCCGGGAGGAAATGACCAAGAATCAAGTCTCCCTGACTTGCCTTGTGAAGGGTTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCAAACGGGCAGCCGGAAAACAACTAGAAGACCACACCTCCGGTGCTGGATTCCGACGGCTCCTTCTTCTTGTACTCGAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCACAACCACTACACTCAGAAGTCGCTCTCGCTGAGCCCCGGGAAG LCDR1 (Combined) 31RASQDISNYLN LCDR2 (Combined) 20 YTSRLQS LCDR3 (Combined) 21 QQGNTLPYTLCDR1 (Kabat) 31 RASQDISNYLN LCDR2 (Kabat) 20 YTSRLQS LCDR3 (Kabat) 21QQGNTLPYT LCDR1 (Chothia) 22 SQDISNY LCDR2 (Chothia) 23 YTSLCDR3 (Chothia) 24 GNTLPY LCDR1 (IMGT) 25 QDISNY LCDR2 (IMGT) 23 YTSLCDR3 (IMGT) 21 QQGNTLPYT VL 32EIVMTQSPATLSLSPGERATLSGRASQDISNYLNWYQQKPGQAPRLLIYYTSRLQSGIPARFSGSGSGADYTLTISSLQPEDFAVYFCQQ GNTLPYTFGQGTKLEIKDNA VL (vector 1) 33 GAGATCGTGATGACCCAGTCCCCTGCGACCCTGTCCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCCGGGCCTCCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCTACTACACCTCCCGGCTGCAGTCCGGCATCCCTGCCAGATTCTCCGGCTCTGGCTCTGGCGCCGACTACACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCGTGTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAA ATCAAG DNA VL (vector 2)40 GAAATCGTGATGACTCAGTCCCCCGCCACTCTCTCGCTGTCCCCTGGCGAACGGGCCACCGTGTCGTGCCGGGCGTCGCAGGACATCTCAAACTATCTGAACTGGTACCAGCAGAAGCCTGGACAGGCACCCAGGCTCCTGATCTACTACACCTCGCGCCTGCAATCCGGAATCCCAGCCCGCTTCTCCGGTTCCGGCTCCGGCGCTGATTACACCCTCACCATTAGCAGCCTGCAGCCGGAGGACTTCGCCGTGTACTTCTGTCAACAAGGAAACACCCTCCCGTACACATTTGGGCAGGGAACCAAGCTGGAG ATTAAG Light Chain 34EIVMTQSPATLSESPGERATLSCRASQDISNYINWYQQKPGQAPRLLIYYTSRLQSGIPARFSGSGSGADYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DNA Light Chain 35GAGATCGTGATGACCCAGTCCCCTGCCACCCTGTCCCTGAGCCCT (vector 1)GGCGAGAGAGCCACCCTGAGCTGCCGGGCCTCCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCTACTACACCTCCCGGCTGCAGTCCGGCATCCCTGCCAGATTCTCCGGCTCTGGCTCTGGCGCCGACTACACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCGTGTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCTCGCGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAAC CGGGGGGAGTGCDNA Light Chain 41 GAAATCGTGATGACTCAGTCCCCCGCCACTCTCTCCCTGTCCCCT(vector 2) GGCGAACGGGCCACCCTGTCGTGCCGGGCGTCGCAGGACATCTCAAACTATCTGAACTGGTACCAGCAGAAGCCTGGACAGGCACCCAGGCTCCTGATCTACTACACCTCGCGCCTGCAATCCGGAATCCCAGCCCGCTTCTCCGGTTCCGGCTCCGGCGCTGATTACACCCTCACCATTAGCAGCCTGCAGCCGGAGGACTTCGCCGTGTACTTCTGTCAACAAGGAAACACCCTCCCGTACACATTTGGGCAGGGAACCAAGCTGGAGATTAAGCGTACGGTGGCCGCGCCGTCCGTGTTCATCTTCCCTCCTTCTGACGAGCAGCTCAAGAGCGGCACCGCGTCGGTGGTCTGCCTGCTGAACAACTTCTACCCACGGGAGGCCAAGGTCCAGTGGAAAGTGGATAACGCATTGCAGTCGGGAAACTCACAGGAGTCGGTGACCGAACAGGACTCCAAAGACTCAACCTACTCCCTGTCCTCCACTCTTACCCTGTCCAAGGCGGACTACGAAAAGCACAAGGTCTACGCCTGCGAAGTGACCCATCAGGGTCTGAGCAGCCCTGTGACTAAGAGCTTTAAC CGCGGCGAATGC

TABLE 2 FGF21 miMetic antibodies SEQ ID NO: Amino Acid Sequence NOV004HCDR1 (Combined) 6 GYSITSGYTWH HCDR2 (Combined) 42 YIHYSVYTNYNPSVKGHCDR3 (Combined) 8 RTTSLERYFDV HCDR1 (Kabat) 9 SGYTWH HCDR2 (Kabat) 42YIHYSVYTNYNPSVKG HCDR3 (Kabat) 8 RTTSLERYFDV HCDR1 (Chothia) 10 GYSITSGYHCDR2 (Chothia) 11 HYSVY HCDR3 (Chothia) 8 RTTSLERYFDV HCDR1 (IMGT) 12GYSITSGYT HCDR2 (IMGT) 13 THYSVYT HCDR3 (IMGT) 14 ARRTTSLERYFDV VH 43EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSS DNA VH 44GAAGTCCAACTCGTCGAATCCGGGGGGGGACTGGTCAAGCCGGGAGGATCGCTGAGACTGTCGTGCGCAGTGTCAGGGTACAGCATCACCTCCGGTTACACCTGGCACTGGGTCAGACAGGCGCCGGGAAAAGGCCTGGAATGGCTGTCCTACATTCATTACTCCGTGTACACTAACTACAACCCCTCAGTGAAGGGGCGGTTCACCATCTCCCGGGACACTGCCAAGAATAGCTTCTATCTGCAAATGAACTCCCTGCGGGCCGAGGATACCGCCGTGTACTACTGCGCGAGGCGCACCACGTCCCTGGAGCGCTACTTTGACGTGTGGGGCCAGGGTACCCTCGTGACTGTGTCCTCG Heavy Chain 45EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWISYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DNA Heavy Chain 46GAAGTCCAACTCGTCGAATCCGGGGGGGGACTGGTCAAGCCGGGAGGATCGCTGAGACTGTCGTGCGCAGTGTCAGGGTACAGCATCACCTCCGGTTACACCTGGCACTGGGTCAGAGAGGCGCCGGGAAAAGGCCTGGAATGGCTGTCCTACATTCATTACTCCGTGTACACTAACTACAACCCCTCAGTGAAGGGGCGGTTCACCATCTCCCGGGACACTGCCAAGAATAGCTTCTATCTGCAAATGAACTCCCTGCGGGCCGAGGATACCGCCGTGTACTACTGCGCGAGGCGCACCACGTCCCTGGAGCGCTACTTTGACGTGTGGGGCCAGGGTACCCTCGTGACTGTGTCCTCGGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGGGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGGGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAAGGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCGTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGCAAG LCDR1 (Combined) 19QASQDISNYLN LCDR2 (Combined) 20 YTSRLQS LCDR3 (Combined) 21 QQGNTLPYTLCDR1 (Kabat) 19 QASQDISNYLN LCDR2 (Kabat) 20 YTSRLQS LCDR3 (Kabat) 21QQGNTLPYT LCDR1 (Chothia) 22 SQDISNY LCDR2 (Chothia) 23 YTSLCDR3 (Chothia) 24 GNTLPY LCDR1 (IMGT) 25 QDISNY LCDR2 (IMGT) 23 YTSLCDR3 (IMGT) 21 QQGNTLPYT VL 47DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQ GNTLPYTFGQGTKLEIK DNA VL48 GATATTCAGATGACTCAGAGCCCCTCCTCGCTCTCCGCCTCCGTGGGGGATCGCGTGACAATCACCTGTCAAGCGTCCCAGGACATCTCAAACTACCTGAACTGGTATCAGCAGAAGCCAGGGAAGGCCCCGAAGCTGCTGATCTACTACACTTCGCGGCTGCAGTCCGGCGTGCCGTCACGGTTCACTGGCTCGGGCTCCGGAGCAGACTACACCTTCACCATTAGCAGCCTGCAGCCCGAGGACATCGCTACCTACTTTTGCCAACAAGGAAACACCCTGCCTTACACCTTCGGACAGGGTACTAAGCTGGAA ATCAAA Light Chain 49DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DNA Light Chain 50GATATTCAGATGACTCAGAGCCCCTCCTCGCTCTCCGCCTCCGTGGGGGATCGCGTGACAATCACCTGTCAAGCGTCCCAGGACATCTCAAACTACCTGAACTGGTATCAGCAGAAGCCAGGGAAGGCCCCGAAGCTGCTGATCTACTACACTTCGCGGCTGCAGTCCGGCGTGCCGTCACGGTTCACTGGCTCGGGCTCCGGAGCAGACTACACCTTCACCATTAGCAGCCTGCAGCCCGAGGACATCGCTACCTACTTTTGCCAACAAGGAAACACCCTGCCTTACACCTTCGGACAGGGTACTAACCTGGAAATCAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NOV001 VH 55QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVILTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSS Heavy Chain 56QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VLDVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQGSHIPYTFGQGTKLEIKLight Chain 58 DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAFSVFIFPPSDEQLKSGTASVVCLLNNFYPREARVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NOV002 VH 43EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSS Heavy Chain 58EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLSYIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRTTSLERYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFFPKPKDTLMISRTPEVTGVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VL 47DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQ GNTLPYTFGQGTKLEIKLight Chain 60 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NOV003 VH 55QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSS Heavy Chain 61QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGRIHPGSGNTYYNEKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAILLLRSYGMDDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSGNVFSCSVMHEALHNHYTQKSLSLSPGK VL 57DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCFQGSHIPYTFGQGTKLEIKLight Chain 62 DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSSGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Other antibodies of the present disclosure include those where the aminoacids or nucleic acids encoding the amino acids have been mutated, yethave at least 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to thesequences described in Table 1. Some embodiments include mutant aminoacid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids havebeen mutated in the variable regions when compared with the variableregions depicted in the sequence described in Table 1, while retainingsubstantially the same antigen-binding activity.

Other antibodies of the present disclosure include those where the aminoacids or nucleic acids encoding the amino acids have been mutated, yethave at least 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to thesequences described in Table 1. Some embodiments include mutant aminoacid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids havebeen mutated in the variable regions when compared with the variableregions depicted in the sequence described in Table 1, while retainingsubstantially the same antigen-binding activity.

Since each of these antibodies can bind to β-klotho, the VH, VL, fulllength light chain, and full length heavy chain sequences (amino acidsequences and the nucleotide sequences encoding the amino acidsequences) can be “mixed and matched” to create other β-klotho-bindingantibodies of the present disclosure. Such “mixed and matched”-β-klothobinding antibodies can be tested using the binding assays known in theart (e.g., ELISAs, and other assays described in the Example section).When these chains are mixed and matched, a VH sequence from a particularVH/VL pairing should be replaced with a structurally similar VHsequence. Likewise a full length heavy chain sequence from a particularfull length heavy chain/full length light chain pairing should bereplaced with a structurally similar full length heavy chain sequence.Likewise, a VL sequence from a particular VH/VL pairing should bereplaced with a structurally similar VL sequence. Likewise a full lengthlight chain sequence from a particular full length heavy chain/fulllength light chain pairing should be replaced with a structurallysimilar full length light chain sequence.

Accordingly, in one aspect, the present disclosure provides an isolatedantibody (e.g., monoclonal antibody) or antigen-binding region thereofhaving: a heavy chain variable domain comprising an amino acid sequenceof SEQ ID NO: 15, and a light chain variable domain comprising an aminoacid sequence of SEQ ID NO: 26 or 32, wherein the antibody specificallybinds to β-klotho (e.g., human and cynomolgus monkey β-klotho).

In another aspect, the present disclosure provides (i) an isolatedantibody having: a full length heavy chain comprising an amino acidsequence, that has been optimized for expression in a mammalian cell, ofSEQ ID NO: 17, and a full length light chain comprising an amino acidsequence, that has been optimized for expression in a mammalian cell, ofSEQ ID NO: 28 or 34; or (ii) a functional protein comprising anantigen-binding portion thereof. More specifically, in certain aspects,the present disclosure provides an isolated antibody or antigen-bindingregion thereof comprising a heavy chain and a light chain comprisingamino acid sequences selected from SEQ ID NOs: 17 and 28; or 17 and 34,respectively.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orfragment comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 15 or an amino acid sequence with atleast 90% or 95% identity thereof; and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO: 26 or 32 or anamino acid sequence with at least 90% or 95% identity thereof.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orfragment comprises a VH comprising the amino acid sequence of SEQ ID NO:15.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orfragment comprises a VL comprising the amino acid sequence of SEQ ID NO:26 or 32.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orfragment comprises a(i) a VH comprising the amino acid sequence of SEQID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 26or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 15 and aVL comprising the amino acid sequence of SEQ ID NO: 32.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibodycomprises (i) a heavy chain comprising the amino acid sequence of SEQ IDNO: 17 and a light chain comprising the amino acid sequence of SEQ IDNO: 28, or (ii) a heavy chain comprising the amino acid sequence of SEQID NO: 17 and a light chain comprising the amino acid sequence of SEQ IDNO: 34.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment does not comprise Combined or Kabat CDRs ofantibody NOV004 as set forth in Table 2.

The terms “complementarity determining region,” and “CDR,” as usedherein refer to the sequences of amino acids within antibody variableregions which confer antigen specificity and binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region(LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme),and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7,132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77(2003) (“IMGT” numbering scheme). For example, for classic formats,under Kabat, the CDR amino acid residues in the heavy chain variabledomain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102(HCDR3); and the CDR amino acid residues in the light chain variabledomain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97(LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32(HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residuesin VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Bycombining the CDR definitions of both Kabat and Chothia, the CDRsconsist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102(HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56(LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acidresidues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2)and 93-102 (CDR3), and the CDR amino acid residues in the VL arenumbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3)(numbering according to “Kabat”). Under IMGT, the CDR regions of anantibody can be determined using the program IMGT/DomainGap Align. Bycombining the CDR definitions of both Kabat and Chothia, the “Combined”CDRs may consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2),and 99-108 (HCDR3) in human VH and amino acid residues 24-39 (LCDR1),55-61 (LCDR2), and 94-102 (LCDR3) in human VL.

In another aspect, the present disclosure provides β-klotho bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2s,and CDR3s as described in Table 1, or combinations thereof. In specificaspects, these CDRs are delineated using the Kabat system. In anotherspecific aspects, these CDRs are delineated using the Combined system.

Given that each of these antibodies can bind to β-klotho and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and matched, although each antibody preferably contains a VH CDR1,2 and 3 and a VL CDR1, 2 and 3 to create other β-klotho bindingmolecules of the present disclosure. Such “mixed and matched” β-klothobinding antibodies can be tested using the binding assays known in theart and those described in the Examples (e.g., ELISAs, SET, Biacore®binding assays). When VH CDR sequences are mixed and matched, the CDR1,CDR2 and/or CDR3 sequence from a particular VH sequence should bereplaced with a structurally similar CDR sequence(s). Likewise, when VLCDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular VL sequence should be replaced with a structurallysimilar CDR sequence(s). It will be readily apparent to the ordinarilyskilled artisan that novel VH and VL sequences can be created bysubstituting one or more VH and/or VL CDR region sequences withstructurally similar sequences from the CDR sequences shown herein formonoclonal antibodies of the present disclosure. In addition to theforegoing, in one embodiment, the antigen-binding fragments of theantibodies described herein can comprise a VH CDR1, 2, and 3, or a VLCDR 1, 2, and 3, wherein the fragment binds to β-klotho as a singlevariable domain.

In certain embodiments of the present disclosure, the antibodies orantigen-binding fragments thereof may have the heavy and light chainsequences of the Fabs described in Table 1. More specifically, theantibody or antigen-binding fragments thereof may have the heavy andlight sequence of Fab NOV005 or NOV006.

In certain embodiments of the present disclosure, the antibody orantigen-binding fragment that specifically binds β-klotho comprisesheavy chain variable region CDR1, CDR2, and CDR3 of Fab NOV005 orNOV006, and light chain variable region CDR1, CDR2, and CDR3 of FabNOV005 or NOV006, for example, as set forth in Table 1.

In other embodiments of the present disclosure the antibody orantigen-binding fragment in that specifically binds β-klotho comprises aheavy chain variable region CDR1, a heavy chain variable region CDR2, aheavy chain variable region CDR3, a light chain variable region CDR1, alight chain variable region CDR2, and a light chain variable region CDR3as defined by Kabat and described in Table 1. In still other embodimentsof the present disclosure the antibody or antigen-binding fragment inthat specifically binds β-klotho comprises a heavy chain variable regionCDR1, a heavy chain variable region CDR2, a heavy chain variable regionCDR3, a light chain variable region CDR1, a light chain variable regionCDR2, and a light chain variable region CDR3 as defined by Chothia anddescribed in Table 1. In still other embodiments of the presentdisclosure the antibody or antigen-binding fragment in that specificallybinds β-klotho comprises a heavy chain variable region CDR1, a heavychain variable region CDR2, a heavy chain variable region CDR3, a lightchain variable region CDR1, a light chain variable region CDR2, and alight chain variable region CDR3 as defined by Combined Kabat andChothia and described in Table 1. In still other embodiments of thepresent disclosure the antibody or antigen-binding fragment in thatspecifically binds β-klotho comprises a heavy chain variable regionCDR1, a heavy chain variable region CDR2, a heavy chain variable regionCDR3, a light chain variable region CDR1, a light chain variable regionCDR2, and a light chain variable region CDR3 as defined by IMGT anddescribed in Table 1.

In certain embodiments, the present disclosure includes antibodies orantigen-binding fragments that specifically bind to β-klotho asdescribed in Table 1, e.g., antibody NOV005 or NOV006. In a preferredembodiment, the antibody, or antigen-binding fragment, that bindsβ-klotho and activates the FGF21 receptor complex is Fab NOV005 orNOV006.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises:

-   -   (i) a heavy chain CDR1 (HCDR1) comprising the amino acid        sequence of SEQ ID NO: 6, 9, 10 or 12;    -   (ii) a heavy chain CDR2 (HCDR2) comprising the amino acid        sequence of SEQ ID NO: 7, 11 or 13;    -   (iii) a heavy chain CDR3 (HCDR3) comprising the amino acid        sequence of SEQ ID NO: 8, or 14;    -   (iv) a light chain CDR1 (LCDR1) comprising the amino acid        sequence of SEQ ID NO: 19, 31, 22, or 25;    -   (v) a light chain CDR2 (LCDR2) comprising the amino acid        sequence of SEQ ID NO: 20 or 23; and    -   (vi) a light chain CDR3 (LCDR3) comprising the amino acid        sequence of SEQ ID NO: 21 or 24.In a specific aspect, provided        herein is an isolated antibody (e.g., monoclonal antibody) or        antigen-binding fragment thereof that binds to an epitope of        β-klotho and induces activity of an FGF21 receptor complex,        e.g., FGFR1c-β-klotho receptor complex, wherein the antibody or        antigen-binding fragment thereof comprises:    -   (i) a heavy chain CDR1 (HCDR1) comprising the amino acid        sequence of SEQ ID NO: 6, 9, 10 or 12;    -   (ii) a heavy chain CDR2 (HCDR2) comprising the amino acid        sequence of SEQ ID NO: 7, 11 or 13;    -   (iii) a heavy chain CDR3 (HCDR3) comprising the amino acid        sequence of SEQ ID NO: 8, or 14;    -   (iv) a light chain CDR1 (LCDR1) comprising the amino acid        sequence of SEQ ID NO: 31, 22, or 25;    -   (v) a light chain CDR2 (LCDR2) comprising the amino acid        sequence of SEQ ID NO: 20 or 23; and    -   (vi) a light chain CDR3 (LCDR3) comprising the amino acid        sequence of SEQ ID NO: 21 or 24.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises:

-   -   (i) a heavy chain CDR1 (HCDR1) comprising the amino acid        sequence of SEQ ID NO: 6, 9, 10 or 12;    -   (ii) a heavy chain CDR2 (HCDR2) comprising the amino acid        sequence of SEQ ID NO: 7, 11 or 13;    -   (iii) a heavy chain CDR3 (HCDR3) comprising the amino acid        sequence of SEQ ID NO: 8, or 14;    -   (iv) a light chain CDR1 (LCDR1) comprising the amino acid        sequence of SEQ ID NO: 19, 31, 22, or 25;    -   (v) a light chain CDR2 (LCDR2) comprising the amino acid        sequence of SEQ ID NO: 20 or 23; and    -   (vi) a light chain CDR3 (LCDR3) comprising the amino acid        sequence of SEQ ID NO: 21 or 24.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, the        HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, the        HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, the        LCDR1 comprises the amino acid sequence of SEQ ID NO: 31, the        LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and        the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   the HCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the        HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, the        HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, the        LCDR1 comprises the amino acid sequence of SEQ ID NO: 31, the        LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and        the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10,        the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11,        the HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, the        LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the        LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and        the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12,        the HCDR2 comprises the amino acid sequence of SEQ ID NO: 13,        the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14,        the LCDR1 comprises the amino acid sequence of SEQ ID NO: 25,        the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23,        and the LCDR3 comprises the amino acid sequence of SEQ ID NO:        21.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   (i) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 6,        the HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, the        HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, the        LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the        LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and        the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21; or    -   (ii) the HCDR1 comprises the amino acid sequence of SEQ ID NO:        9, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 7,        the HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, the        LCDR1 comprises the amino acid sequence of SEQ ID NO: 19, the        LCDR2 comprises the amino acid sequence of SEQ ID NO: 20, and        the LCDR3 comprises the amino acid sequence of SEQ ID NO: 21.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-o-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   the HCDR1 comprises the amino acid sequence of SEQ ID NO: 10,        the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11,        the HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, the        LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the        LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and        the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising HCDR1, HCDR2,and HCDR3, and a VL comprising LCDR1, LCDR2, and LCDR3, wherein:

-   -   the HCDR1 comprising the amino acid sequence of SEQ ID NO: 12,        the HCDR2 comprises the amino acid sequence of SEQ ID NO: 13,        the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14,        the LCDR1 comprises the amino acid sequence of SEQ ID NO: 25,        the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23,        and the LCDR3 comprises the amino acid sequence of SEQ ID NO:        21.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising CDRs HCDR1,HCDR2 and HCDR3 and a VL comprising CDRs LCDR1, LCDR2, and LCDR3,wherein the antibody or antigen-binding fragment thereof comprises: aHCDR1 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR2comprising the amino acid sequence of SEQ ID NO: 7, a HCDR3 comprisingthe amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the aminoacid sequence of SEQ ID NO: 19 or 31, a LCDR2 comprising the amino acidsequence of SEQ ID NO: 20, and a LCDR3 comprising the amino acidsequence of SEQ ID NO: 21.

In a specific aspect, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment thereof comprises a VH comprising CDRs HCDR1,HCDR2 and HCDR3 and a VL comprising CDRs LCDR1, LCDR2, and LCDR3,wherein the antibody or antigen-binding fragment thereof comprises: aHCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a HCDR2comprising the amino acid sequence of SEQ ID NO: 7, a HCDR3 comprisingthe amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the aminoacid sequence of SEQ ID NO: 19 or 31, a LCDR2 comprising the amino acidsequence of SEQ ID NO: 20, and a LCDR3 comprising the amino acidsequence of SEQ ID NO: 21.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor fragment increases the activity of β-klotho and/or FGFR1c. Inspecific aspects, said antibody or fragment thereof increases theactivity of β-klotho and/or FGFR1c, for example as determined byphospho-ERK activity, by at least about 10% or 20%. In specific aspects,said antibody or fragment thereof increases the activity of β-klothoand/or FGFR1c, for example as determined by phospho-ERK activity, by atleast about 30% or 40%. In specific aspects, said antibody or fragmentthereof increases the activity of β-klotho and/or FGFR1c, for example asdetermined by phospho-ERK activity, by at least about 50% or 60%. Inspecific aspects, said antibody or fragment thereof increases theactivity of β-klotho and/or FGFR1c, for example as determined byphospho-ERK activity, by at least about 70% or 80%. In specific aspects,said antibody or fragment thereof increases the activity of β-klothoand/or FGFR1c, for example as determined by phospho-ERK activity, by atleast about 90% or 95%.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment binds to a human β-klotho protein with a K_(D)of less than or equal to 500 pM or 450 pM, for example as determined byBIACORE™ binding assays. In specific aspects, provided herein is anisolated antibody (e.g., monoclonal antibody) or antigen-bindingfragment thereof that binds to an epitope of β-klotho and inducesactivity of an FGF21 receptor complex, e.g., FGFR1c-β-klotho receptorcomplex, wherein the antibody or antigen-binding fragment binds to ahuman β-klotho protein with a K_(D) of less than or equal to 450 pM or400 pM, for example as determined by BIACORE™ binding assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein the antibody orantigen-binding fragment binds to a human β-klotho protein with a K_(D)of less than or equal to 10 pM or 20 pM, for example as determined byBIACORE™ binding assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said epitope ofβ-klotho comprises one or more amino acids of residues 246-265, 536-550,834-857 and 959-986 of the β-klotho sequence (SEQ ID NO:52).

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said epitopescomprises one or more of amino acids of residues 646-670, 696-700, and646-689 of the β-klotho sequence (SEQ ID NO:52).

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment protects one or more amino acids of residues246-265, 536-550, 834-857 and 959-986 of human β-klotho (SEQ ID NO:52),as determined by hydrogen-deuterium exchange (HDx).

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment protects one or more of amino acids ofresidues 646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ IDNO:52).

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor fragment does not contact residues 701 (Tyr) or 703 (Arg) of humanβ-klotho (SEQ ID NO: 52).

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating the humanFGFR1c_β-klotho receptor complex, for example, with an EC50 of less thanor equal to 50 nM, as measured by pERK cell assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating the humanFGFR1c_β-klotho receptor complex, for example, with an EC50 of less thanor equal to 100 nM, as measured by pERK cell assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating the humanFGFR1c_β-klotho receptor complex, for example, with an EC50 of less thanor equal to 40 nM or 30 nM, as measured by pERK cell assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating a humanFGFR1c_β-klotho receptor complex, for example, with an EC50 of less thanor equal to 50 nM, as measured by pERK cell assays, and wherein theantibody or antigen-binding fragment thereof is not capable ofactivating an FGFR2c_β-klotho receptor complex, an FGFR3c_β-klothoreceptor complex, and/or FGFR4_β-klotho receptor complex.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating thecynomolgus monkey FGFR1c_β-klotho receptor complex with an EC50 of lessthan or equal to 50 nM, as measured by pERK cell assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating thecynomolgus monkey FGFR1c_β-klotho receptor complex with an EC50 of lessthan or equal to 40 nM or 30 nM, as measured by pERK cell assays.

In specific aspects, provided herein is an isolated antibody (e.g.,monoclonal antibody) or antigen-binding fragment thereof that binds toan epitope of β-klotho and induces activity of an FGF21 receptorcomplex, e.g., FGFR1c-β-klotho receptor complex, wherein said antibodyor antigen-binding fragment thereof is capable of activating thecynomolgus monkey FGFR1c_β-klotho receptor complex with an EC50 of lessthan or equal to 20 nM, as measured by pERK cell assays. As used herein,a human antibody comprises heavy or light chain variable regions or fulllength heavy or light chains that are “the product of” or “derived from”a particular germline sequence if the variable regions or full lengthchains of the antibody are obtained from a system that uses humangermline immunoglobulin genes. Such systems include immunizing atransgenic mouse carrying human immunoglobulin genes with the antigen ofinterest or screening a human immunoglobulin gene library displayed onphage with the antigen of interest. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody.

A human antibody that is “the product of” or “derived from” a particularhuman germline immunoglobulin sequence may contain amino aciddifferences as compared to the germline sequence, due to, for example,naturally occurring somatic mutations or intentional introduction ofsite-directed mutations. However, in the VH or VL framework regions, aselected human antibody typically is at least 90% identical in aminoacids sequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or99% identical in amino acid sequence to the amino acid sequence encodedby the germline immunoglobulin gene.

Typically, a recombinant human antibody will display no more than 10amino acid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene in the VH or VL framework regions. Incertain cases, the human antibody may display no more than 5, or even nomore than 4, 3, 2, or 1 amino acid difference from the amino acidsequence encoded by the germline immunoglobulin gene. Examples of humangermline immunoglobulin genes include, but are not limited to thevariable domain germline fragments described below, as well as DP47 andDPK9.

Homologous Antibodies

In yet another embodiment, the present disclosure provides an antibody,or an antigen-binding fragment thereof, comprising amino acid sequencesthat are homologous to the sequences described in Table 1, and theantibody binds to a β-klotho protein (e.g., human and cynomolgus monkeyβ-klotho), and retains the desired functional properties (e.g.,activating or increasing the activity of a β-klotho/FGFR1c receptorcomplex and/or one or more activities of FGF21) of those antibodiesdescribed in Table 1.

For example, the present disclosure provides an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex), or a functional antigen-bindingfragment thereof, comprising a heavy chain variable domain and a lightchain variable domain, wherein the heavy chain variable domain comprisesan amino acid sequence that is at least 80%, at least 90%, 95%, 96%,97%, 98%, or at least 99% identical to an amino acid sequence of SEQ IDNO: 15; the light chain variable domain comprises an amino acid sequencethat is at least 80%, at least 90%, 95%, 96%, 97%, 98%, or at least 99%identical to an amino acid sequence of SEQ ID NO: 26 or 32; wherein theantibody specifically binds to β-klotho (e.g., human and cynomolgusmonkey β-klotho) and induces activity of β-klotho, and wherein the heavychain variable domain does not comprise an amino acid sequence set forthin Table 2, e.g., the amino acid sequence of SEQ ID NO: 43 or 55 and thelight chain variable region does not comprise an amino acid sequence setforth in Table 2, e.g., the amino acid sequence of SEQ ID NO: 47 or 57.In certain aspects of the present disclosure the heavy and light chainsequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3sequences as defined by Kabat, for example as set forth in Table 1. Incertain other aspects of the present disclosure the heavy and lightchain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, andLCDR3 sequences as defined by Chothia, for example as set forth in Table1, respectively. In certain other aspects of the present disclosure theheavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3 sequences as defined by Combined, for example asset forth in Table 1, respectively. In certain other aspects of thepresent disclosure the heavy and light chain sequences further compriseHCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined byIMGT, for example as set forth in Table 1, respectively.

For example, the present disclosure provides an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex), or a functional antigen-bindingfragment thereof, comprising a heavy chain variable domain and a lightchain variable domain, wherein the heavy chain variable domain comprisesan amino acid sequence that is at least 80%, at least 90%, 95%, 96%,97%, 98%, or at least 99% identical to an amino acid sequence of SEQ IDNos: 15; the light chain variable domain comprises an amino acidsequence that is at least 80%, at least 90%, 95%, 96%, 97%, 98%, or atleast 99% identical to an amino acid sequence of SEQ ID Nos: 26 or 32;wherein the antibody specifically binds to β-klotho (e.g., human andcynomolgus monkey β-klotho), and wherein the heavy chain variable domaindoes not comprise an amino acid sequence set forth in Table 2, e.g., theamino acid sequence of SEQ ID NO: 43 or 55 and the light chain variableregion does not comprise an amino acid sequence set forth in Table 2,e.g., the amino acid sequence of SEQ ID NO: 47 or 57. In certain aspectsof the present disclosure the heavy and light chain sequences furthercomprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences asdefined by Kabat, for example as set forth in Table 1. In certain otheraspects of the present disclosure the heavy and light chain sequencesfurther comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequencesas defined by Chothia, for example as set forth in Table 1. In certainother aspects of the present disclosure the heavy and light chainsequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3sequences as defined by Combined, for example as set forth in Table 1.In certain other aspects of the present disclosure the heavy and lightchain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, andLCDR3 sequences as defined by IMGT, for example as set forth in Table 1.

In other embodiments, the VH and/or VL amino acid sequences may be 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequencesset forth in Table 1, wherein the heavy chain variable domain does notcomprise an amino acid sequence set forth in Table 2, e.g., the aminoacid sequence of SEQ ID NO: 43 or 55 and the light chain variable regiondoes not comprise an amino acid sequence set forth in Table 2, e.g., theamino acid sequence of SEQ ID NO: 47 or 57. In other embodiments, the VHand/or VL amino acid sequences may be identical except for an amino acidsubstitution in no more than 1, 2, 3, 4 or 5 amino acid positions. Anantibody having VH and VL regions having high (i.e., 80% or greater)identity to the VH and VL regions of those described in Table 1 can beobtained by mutagenesis (e.g., site-directed or PCR-mediatedmutagenesis) of nucleic acid molecules encoding SEQ ID Nos: 10, 30, 50,or 70 and SEQ ID Nos: 20, 40, 60, or 80, respectively, followed bytesting of the encoded altered antibody for retained function using thefunctional assays described herein, wherein the heavy chain variabledomain does not comprise an amino acid sequence set forth in Table 2,e.g., the amino acid sequence of SEQ ID NO: 43 or 55 and the light chainvariable region does not comprise an amino acid sequence set forth inTable 2, e.g., the amino acid sequence of SEQ ID NO: 47 or 57.

In other embodiments, the full length heavy chain and/or full lengthlight chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to the sequences set forth in Table 1,wherein the heavy chain does not comprise a heavy chain amino acidsequence set forth in Table 2, e.g., the amino acid sequence of SEQ IDNO: 45, 56, 59, or 61, and the light chain does not comprise a lightchain amino acid sequence set forth in Table 2, e.g., the amino acidsequence of SEQ ID NO: 49, 58, 60, or 62. An antibody having a fulllength heavy chain and full length light chain having high (i.e., 80% orgreater) identity to the full length heavy chain of SEQ ID NO: 17, andfull length light chain of SEQ ID NO: 28 or 34, can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleicacid molecules encoding such polypeptides, followed by testing of theencoded altered antibody for retained function using the functionalassays described herein, wherein the heavy chain does not comprise aheavy chain amino acid sequence set forth in Table 2, e.g., the aminoacid sequence of SEQ ID NO: 45, 56, 59, or 61, and the light chain doesnot comprise a light chain amino acid sequence set forth in Table 2,e.g., the amino acid sequence of SEQ ID NO: 49, 58, 60, or 62. Anantibody having a full length heavy chain and full length light chainhaving high (i.e., 80% or greater) identity to the full length heavychain of SEQ ID NO: 17, and full length light chain of SEQ ID NO: 28 or34, can be obtained by mutagenesis (e.g., site-directed or PCR-mediatedmutagenesis) of nucleic acid molecules encoding such polypeptides,followed by testing of the encoded altered antibody for retainedfunction using the functional assays described herein, wherein the heavychain does not comprise a heavy chain amino acid sequence set forth inTable 2, e.g., the amino acid sequence of SEQ ID NO: 45, 56, 59, or 61,and the light chain does not comprise a light chain amino acid sequenceset forth in Table 2, e.g., the amino acid sequence of SEQ ID NO: 49,58, 60, or 62.

In other embodiments, the full length heavy chain and/or full lengthlight chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequences set forth in Table 1, whereinthe heavy chain does not comprise a heavy chain amino acid sequence setforth in Table 2, e.g., the amino acid sequence of SEQ ID NO: 45, 56,59, or 61, and the light chain does not comprise a light chain aminoacid sequence set forth in Table 2, e.g., the amino acid sequence of SEQID NO: 49, 58, 60, or 62.

In other embodiments, the variable regions of heavy chain and/or thevariable regions of light chain nucleotide sequences may be 60%, 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forthin Table 1, wherein the heavy chain does not comprise a heavy chainamino acid sequence set forth in Table 2, e.g., the amino acid sequenceof SEQ ID NO: 45, 56, 59, or 61, and the light chain does not comprise alight chain amino acid sequence set forth in Table 2, e.g., the aminoacid sequence of SEQ ID NO: 49, 58, 60, or 62.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity equals number of identical positions/total number ofpositions ×100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the presentdisclosure can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.For example, such searches can be performed using the BLAST program(version 2.0) of Altschul, et al., 1990 J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the present disclosure has aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencesand a light chain variable region comprising CDR1, CDR2, and CDR3sequences, wherein one or more of these CDR sequences have specifiedamino acid sequences based on the antibodies described herein orconservative modifications thereof, and wherein the antibodies retainthe desired functional properties of the β-klotho-binding antibodies ofthe present disclosure.

Accordingly, the present disclosure provides an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex), or a antigen-binding fragmentthereof, consisting of a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences, wherein: the heavy chain variable regionCDR1 amino acid sequences are selected from the group consisting of SEQID NO: 6, and conservative modifications thereof; the heavy chainvariable region CDR2 amino acid sequences are selected from the groupconsisting of SEQ ID NO: 7, and conservative modifications thereof; theheavy chain variable region CDR3 amino acid sequences are selected fromthe group consisting of SEQ ID NO: 8, and conservative modificationsthereof; the light chain variable regions CDR1 amino acid sequences areselected from the group consisting of SEQ ID Nos: 19 and 31, andconservative modifications thereof, the light chain variable regionsCDR2 amino acid sequences are selected from the group consisting of SEQID NO: 20, and conservative modifications thereof; the light chainvariable regions of CDR3 amino acid sequences are selected from thegroup consisting of SEQ ID NO: 21, and conservative modificationsthereof, and the antibody or antigen-binding fragments thereofspecifically binds to β-klotho.

Accordingly, the present disclosure provides an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex), or a antigen-binding fragmentthereof, consisting of a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences, wherein: the heavy chain variable regionCDR1 amino acid sequences are selected from the group consisting of SEQID Nos: 6, 9, 10, and 12, and conservative modifications thereof; theheavy chain variable region CDR2 amino acid sequences are selected fromthe group consisting of SEQ ID Nos: 7, 11, and 13, and conservativemodifications thereof; the heavy chain variable region CDR3 amino acidsequences are selected from the group consisting of SEQ ID Nos: 8 and14, and conservative modifications thereof; the light chain variableregions CDR1 amino acid sequences are selected from the group consistingof SEQ ID Nos: 19, 31, 22, and 25, and conservative modificationsthereof; the light chain variable regions CDR2 amino acid sequences areselected from the group consisting of SEQ ID Nos: 20 and 23, andconservative modifications thereof; the light chain variable regions ofCDR3 amino acid sequences are selected from the group consisting of SEQID Nos: 21 or 24, and conservative modifications thereof, and theantibody or antigen-binding fragments thereof specifically binds toβ-klotho.

In one aspect, the present disclosure provides an isolated antibodyoptimized for expression in a mammalian cell comprising a heavy chainvariable region and a light chain variable region wherein the heavychain variable region has amino acid sequences selected from the groupof SEQ ID NO: 15, and conservative modifications thereof, and the fulllength light chain has amino acid sequences selected from the group ofSEQ ID Nos: 26 and 32, and conservative modifications thereof, and theantibody specifically binds to β-klotho (e.g., human and cynomolgusmonkey β-klotho), and wherein the heavy chain variable region does notcomprise a heavy chain variable region amino acid sequence set forth inTable 2, e.g., the amino acid sequence of SEQ ID NO: 43 or 55 and thelight chain variable region does not comprise a light chain variableregion amino acid sequence set forth in Table 2, e.g., the amino acidsequence of SEQ ID NO: 47 or 57.

In other embodiments, the antibody of the present disclosure isoptimized for expression in a mammalian cell has a full length heavychain sequence and a full length light chain sequence, wherein one ormore of these sequences have specified amino acid sequences based on theantibodies described herein or conservative modifications thereof, andwherein the antibodies retain the desired functional properties of theβ-klotho binding antibodies of the present disclosure. Accordingly, thepresent disclosure provides an isolated antibody optimized forexpression in a mammalian cell consisting of a full length heavy chainand a full length light chain wherein the full length heavy chain hasamino acid sequences selected from the group of SEQ ID NO: 17, andconservative modifications thereof, and the full length light chain hasamino acid sequences selected from the group of SEQ ID Nos: 28 and 34,and conservative modifications thereof, and the antibody specificallybinds to β-klotho (e.g., human and cynomolgus monkey β-klotho), andwherein the heavy chain does not comprise a heavy chain amino acidsequence set forth in Table 2, e.g., the amino acid sequence of SEQ IDNO: 45, 56, 59, or 61, and the light chain does not comprise a lightchain amino acid sequence set forth in Table 2, e.g., the amino acidsequence of SEQ ID NO: 49, 58, 60, or 62.

Antibodies that Bind to the Same Epitope(s) or that Compete for Bindingto the Same Epitope(s)

The present disclosure provides antibodies (e.g., antibodies capable ofactivating or increasing the activity of a β-klotho/FGFR1c receptorcomplex) that bind to the same epitope as the β-klotho bindingantibodies described in Table 1 (e.g., NOV005 or NOV006). In aparticular aspect, such antibodies and antigen-binding fragments arecapable of increasing the activity of β-klotho and FGFR1c. Additionalantibodies can therefore be identified based on their ability to compete(e.g., to competitively inhibit the binding of, in a statisticallysignificant manner) with other antibodies of the present disclosure inβ-klotho binding assays (such as those described in the Examples). Theability of a test antibody to inhibit the binding of antibodies of thepresent disclosure to a β-klotho protein demonstrates that the testantibody can compete with that antibody for binding to β-klotho; such anantibody may, according to non-limiting theory, bind to the same or arelated (e.g., a structurally similar or spatially proximal) epitope onthe β-klotho protein as the antibody with which it competes. In acertain embodiment, the antibody that binds to the same epitope onβ-klotho as the antibodies of the present disclosure is a humanmonoclonal antibody. Such human monoclonal antibodies can be preparedand isolated as described herein. As used herein, an antibody “competes”for binding when the competing antibody inhibits β-klotho binding of anantibody or antigen-binding fragment of the present disclosure by morethan 50% (for example, 80%, 85%, 90%, 95%, 98% or 99%) in the presenceof an equimolar concentration of competing antibody. In a certainembodiment, the antibody that binds to the same epitope on β-klotho asthe antibodies of the present disclosure is a humanized monoclonalantibody. Such humanized monoclonal antibodies can be prepared andisolated as described herein.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to the same epitope asβ-klotho binding antibody NOV005 or NOV006.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to the same epitope as aβ-klotho binding antibody which comprises a heavy chain variable region(VH) comprising the amino acid sequence of SEQ ID NO: 15 and a lightchain variable region (VL) comprising the amino acid sequence of SEQ IDNO: 26 or 32.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to the same epitope as aβ-klotho binding antibody which comprises a heavy chain variable region(VH) comprising the amino acid sequence of SEQ ID NO: 15 and a lightchain variable region (VL) comprising the amino acid sequence of SEQ IDNO: 26.

In a particular aspect, the present disclosure provides antibodies thatbind to the same epitope as β-klotho binding antibody which comprises aheavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 15 and a light chain variable region (VL) comprising theamino acid sequence of SEQ ID NO: 32.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to an overlapping epitope asβ-klotho binding antibody NOV005 or NOV006.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to an overlapping epitope asa β-klotho binding antibody which comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 15 and alight chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 26 or 32.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to an overlapping epitope asa β-klotho binding antibody which comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 15 and alight chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 26.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that bind to an overlapping epitope asa β-klotho binding antibody which comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 15 and alight chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 32.

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprises oneor more amino acids of residues 246-265 of the β-klotho sequence (SEQ IDNO: 52). In a particular aspect, provided herein is an isolated antibody(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprises oneor more amino acids of residues 536-550 of the β-klotho sequence (SEQ IDNO:52). In a particular aspect, provided herein is an isolated antibody(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprises oneor more amino acids of residues 834-857 of the β-klotho sequence (SEQ IDNO:52). In a particular aspect, provided herein is an isolated antibody(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1 c receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprises oneor more amino acids of residues 959-986 of the β-klotho sequence (SEQ IDNO:52).

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprises oneor more amino acids of residues 246-265, 536-550, 834-857 and 959-986 ofthe β-klotho sequence (SEQ ID NO:52). In specific aspects, providedherein is an isolated antibody (e.g., antibody capable of activating orincreasing the activity of a β-klotho/FGFR1c receptor complex) orantigen-binding fragment thereof that binds to an epitope of β-klotho,wherein said epitope comprises two or more amino acids of residues246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ IDNO:52). In specific aspects, provided herein is an isolated antibody(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprisesthree or more amino acids of residues 246-265, 536-550, 834-857 and959-986 of the β-klotho sequence (SEQ ID NO:52). In specific aspects,provided herein is an isolated antibody (e.g., antibody capable ofactivating or increasing the activity of a β-klotho/FGFR1c receptorcomplex) or antigen-binding fragment thereof that binds to an epitope ofβ-klotho, wherein said epitope comprises amino acids of residues246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ IDNO:52). In specific aspects, provided herein is an isolated antibody(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFRic receptor complex) or antigen-binding fragment thereofthat binds to an epitope of β-klotho, wherein said epitope comprises atleast one amino acid residue from each of the following stretches ofamino acid residues: 246-265, 536-550, 834-857 and 959-986 of theβ-klotho sequence (SEQ ID NO:52).

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to one or more epitopes of β-klotho, wherein said epitopescomprises one or more of amino acids of residues 646-670 of the β-klothosequence (SEQ ID NO:52). In a particular aspect, provided herein is anisolated antibody (e.g., antibody capable of activating or increasingthe activity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to one or more epitopes of β-klotho, whereinsaid epitopes comprises one or more of amino acids of residues 696-700of the β-klotho sequence (SEQ ID NO:52). In a particular aspect,provided herein is an isolated antibody (e.g., antibody capable ofactivating or increasing the activity of a β-klotho/FGFR1c receptorcomplex) or antigen-binding fragment thereof that binds to one or moreepitopes of β-klotho, wherein said epitopes comprises one or more ofamino acids of residues 646-689 of the β-klotho sequence (SEQ ID NO:52).

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to one or more epitopes (e.g., discontinuous epitopes) ofβ-klotho, wherein said epitopes comprises one, or two, or three, orfour, or five, or more of amino acids of residues 646-670, 696-700, and646-689 of the β-klotho sequence (SEQ ID NO:52). In a certain aspect,provided herein is an isolated antibody (e.g., antibody capable ofactivating or increasing the activity of a β-klotho/FGFR1c receptorcomplex) or antigen-binding fragment thereof that binds to two or moreepitopes (e.g., discontinuous epitopes) of β-klotho, wherein saidepitopes comprises one or more of amino acids of residues 646-670,696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:52). In aspecific aspect, provided herein is an isolated antibody (e.g., antibodycapable of activating or increasing the activity of a β-klotho/FGFR1creceptor complex) or antigen-binding fragment thereof that binds tothree or more epitopes (e.g., discontinuous epitopes) of β-klotho,wherein said epitopes comprises one or more of amino acids of residues646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:52).In a specific aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGF1tic receptor complex) or antigen-binding fragment thereofthat binds to three or more epitopes (e.g., discontinuous epitopes) ofβ-klotho, wherein said epitopes comprises amino acids of residues646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:52).In a specific aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGF1tic receptor complex) or antigen-binding fragment thereofthat binds to three or more epitopes (e.g., discontinuous epitopes) ofβ-klotho, wherein said epitopes comprises amino acid residues from eachof the following ranges of amino acid residues: 646-670, 696-700, and646-689 of the β-klotho sequence (SEQ ID NO:52).

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat protects, as determined by hydrogen-deuterium exchange (HDx), one,two, three, four, five, or more of the following peptides of β-klotho(SEQ ID NO: 52): amino acid residues 245-266, 246-265, 343-349, 344-349,421-429, 488-498, 509-524, 536-550, 568-576, 646-669, 646-670, 696-700,773-804, 834-857, and 959-986.

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat protects, as determined by hydrogen-deuterium exchange (HDx), thefollowing peptides of β-klotho (SEQ ID NO: 52): amino acid residues246-265, 536-550, 834-857 and 959-986.

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat protects, as determined by hydrogen-deuterium exchange (HDx), thefollowing peptides of β-klotho (SEQ ID NO: 52): amino acid residues245-266, 246-265, 343-349, 344-349, 421-429, 488-498, 509-524, 536-550,568-576, 646-669, 646-670, 696-700, 773-804, 834-857, and 959-986.

In certain aspects, provided herein is isolated antibody (e.g., antibodycapable of activating or increasing the activity of a β-klotho/FGFR1creceptor complex) or antigen-binding fragment thereof, which increasesthe activity of β-klotho and FGFR1c, wherein the antibody orantigen-binding fragment thereof does not contact residues 701 (Tyr) or703 (Arg) of human β-klotho (SEQ ID NO: 52).

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to β-klotho, wherein said antibody or antigen-bindingfragment thereof contacts one or more amino acids of residues 246-265 ofthe β-klotho sequence (SEQ ID NO: 52), for example as determined byx-ray crystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis. In a particular aspect, provided herein is an isolatedantibody (e.g., antibody capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofcontacts one or more amino acids of residues 536-550 of the β-klothosequence (SEQ ID NO:52), for example as determined by x-raycrystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis. In a particular aspect, provided herein is an isolatedantibody (e.g., antibody capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody orantigen-binding fragment thereof contacts one or more amino acids ofresidues 834-857 of the β-klotho sequence (SEQ ID NO:52), for example asdetermined by x-ray crystallography, hydrogen-deuterium exchange assay,or scanning mutagenesis. In a particular aspect, provided herein is anisolated antibody (e.g., antibody capable of activating or increasingthe activity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody orantigen-binding fragment thereof contacts one or more amino acids ofresidues 959-986 of the β-klotho sequence (SEQ ID NO:52), for example asdetermined by x-ray crystallography, hydrogen-deuterium exchange assay,or scanning mutagenesis.

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to β-klotho, wherein said antibody or antigen-bindingfragment thereof contacts one or more amino acids of residues 246-265,536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ ID NO:52),for example as determined by x-ray crystallography, hydrogen-deuteriumexchange assay, or scanning mutagenesis. In specific aspects, providedherein is an isolated antibody (e.g., antibody capable of activating orincreasing the activity of a β-klotho/FGFR1c receptor complex) orantigen-binding fragment thereof that binds to β-klotho, wherein saidantibody or antigen-binding fragment thereof contacts two or more aminoacids of residues 246-265, 536-550, 834-857 and 959-986 of the β-klothosequence (SEQ ID NO:52), for example as determined by x-raycrystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis. In specific aspects, provided herein is an isolatedantibody (e.g., antibody capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody orantigen-binding fragment thereof contacts three or more amino acids ofresidues 246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence(SEQ ID NO:52), for example as determined by x-ray crystallography,hydrogen-deuterium exchange assay, or scanning mutagenesis. In specificaspects, provided herein is an isolated antibody (e.g., antibody capableof activating or increasing the activity of a β-klotho/FGFR1c receptorcomplex) or antigen-binding fragment thereof that binds β-klotho,wherein said antibody or antigen-binding fragment thereof contacts aminoacids of residues 246-265, 536-550, 834-857 and 959-986 of the β-klothosequence (SEQ ID NO:52), for example as determined by x-raycrystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis. In specific aspects, provided herein is an isolatedantibody (e.g., antibody capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds β-klotho, wherein said antibody orantigen-binding fragment thereof contacts at least one amino acidresidue from each of the following stretches of amino acid residues:246-265, 536-550, 834-857 and 959-986 of the β-klotho sequence (SEQ IDNO:52), for example as determined by x-ray crystallography,hydrogen-deuterium exchange assay, or scanning mutagenesis.

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to β-klotho, wherein said antibody or antigen-bindingfragment thereof contacts one or more of amino acids of residues 646-670of the β-klotho sequence (SEQ ID NO:52), for example as determined byx-ray crystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis. In a particular aspect, provided herein is an isolatedantibody (e.g., antibody capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody orantigen-binding fragment thereof contacts one or more of amino acids ofresidues 696-700 of the β-klotho sequence (SEQ ID NO:52), for example asdetermined by x-ray crystallography, hydrogen-deuterium exchange assay,or scanning mutagenesis. In a particular aspect, provided herein is anisolated antibody (e.g., antibody capable of activating or increasingthe activity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody orantigen-binding fragment thereof contacts one or more of amino acids ofresidues 646-689 of the β-klotho sequence (SEQ ID NO:52), for example asdetermined by x-ray crystallography, hydrogen-deuterium exchange assay,or scanning mutagenesis.

In a particular aspect, provided herein is an isolated antibody (e.g.,antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) or antigen-binding fragment thereofthat binds to β-klotho, wherein said antibody or antigen-bindingfragment thereof contacts one, or two, or three, or four, or five, ormore of amino acids of residues 646-670, 696-700, and 646-689 of theβ-klotho sequence (SEQ ID NO:52), for example as determined by x-raycrystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis. In a certain aspect, provided herein is an isolatedantibody (e.g., antibody capable of activating or increasing theactivity of a β-klotho/FGFR1c receptor complex) or antigen-bindingfragment thereof that binds to β-klotho, wherein said antibody orantigen-binding fragment thereof contacts one or more of the amino acidresidues from each of the following stretches of amino acid residues646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ ID NO:52),for example as determined by x-ray crystallography, hydrogen-deuteriumexchange assay, or scanning mutagenesis. In a specific aspect, providedherein is an isolated antibody (e.g., antibody capable of activating orincreasing the activity of a β-klotho/FGFR1c receptor complex) orantigen-binding fragment thereof that binds to β-klotho, wherein saidantibody or antigen-binding fragment thereof contacts one or more of theamino acid residues from each of the following stretches of amino acidresidues 646-670, 696-700, and 646-689 of the β-klotho sequence (SEQ IDNO:52), for example as determined by x-ray crystallography,hydrogen-deuterium exchange assay, or scanning mutagenesis. In aspecific aspect, provided herein is an isolated antibody (e.g., antibodycapable of activating or increasing the activity of a β-klotho/FGFR1creceptor complex) or antigen-binding fragment thereof that binds toβ-klotho, wherein said antibody or antigen-binding fragment thereofcontacts amino acid residues 646-670, 696-700, and 646-689 of theβ-klotho sequence (SEQ ID NO:52), for example as determined by x-raycrystallography, hydrogen-deuterium exchange assay, or scanningmutagenesis.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that compete with antibody NOV005 orNOV006 for binding to β-klotho.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that compete for binding to β-klothowith a β-klotho binding antibody which comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 15 and alight chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 26 or 32.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that compete for binding to β-klothowith a β-klotho binding antibody which comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 15 and alight chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 26.

In a particular aspect, the present disclosure provides antibodies(e.g., antibody capable of activating or increasing the activity of aβ-klotho/FGFR1c receptor complex) that compete for binding to β-klothowith a β-klotho binding antibody which comprises a heavy chain variableregion (VH) comprising the amino acid sequence of SEQ ID NO: 15 and alight chain variable region (VL) comprising the amino acid sequence ofSEQ ID NO: 32.

Engineered and Modified Antibodies

An antibody (e.g., NOV005 or NOV006) of the present disclosure furthercan be prepared using an antibody having one or more of the VH and/or VLsequences shown herein as starting material to engineer a modifiedantibody, which modified antibody may have altered properties from thestarting antibody. An antibody can be engineered by modifying one ormore residues within one or both variable regions (i. e., VH and/or VL),for example within one or more CDR regions and/or within one or moreframework regions. Additionally or alternatively, an antibody can beengineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.).

Accordingly, another embodiment of the present disclosure pertains to anisolated antibody, or an antigen-binding fragment thereof, comprising aheavy chain variable region comprising CDR1 sequences having an aminoacid sequence selected from the HCDR1 sequences set forth in Table 1;CDR2 sequences having an amino acid sequence selected from the HCDR2sequences set forth in Table 1; CDR3 sequences having an amino acidsequence selected from the HCDR3 sequences set forth in table 1; and alight chain variable region having CDR1 sequences having an amino acidsequence selected from the LCDR1 sequences set forth in Table 1; CDR2sequences having an amino acid sequence selected from the LCDR2sequences set forth in Table 1; and CDR3 sequences consisting of anamino acid sequence selected from the LCDR3 sequences set forth inTable 1. Thus, such antibodies contain the VH and VL CDR sequences ofmonoclonal antibodies, yet may contain different framework sequencesfrom these antibodies.

Accordingly, another embodiment of the present disclosure pertains to anisolated antibody, or an antigen-binding fragment thereof, comprising aheavy chain variable region comprising CDR1 sequences comprising anamino acid sequence selected from the group consisting of SEQ ID Nos: 6and 9; CDR2 sequences comprising an amino acid sequence of SEQ ID NO: 7;CDR3 sequences comprising an amino acid sequence of SEQ ID NO: 8; and alight chain variable region having CDR1 sequences comprising an aminoacid sequence selected from the group consisting of SEQ ID Nos: 19 and31; CDR2 sequences comprising an amino acid sequence of SEQ ID NO: 20;and CDR3 sequences comprising of an amino acid sequence of SEQ ID NO:21. Thus, such antibodies contain the VH and VL CDR sequences ofmonoclonal antibodies, yet may contain different framework sequencesfrom these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the world wide web at mrc-cpe.cam.ac.uk/vbase),as well as in Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.,1992 J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

An example of framework sequences for use in the antibodies of thepresent disclosure are those that are structurally similar to theframework sequences used by selected antibodies of the presentdisclosure, e.g., consensus sequences and/or framework sequences used bymonoclonal antibodies of the present disclosure. The VH CDR1, 2 and 3sequences, and the VL CDR1, 2 and 3 sequences, can be grafted ontoframework regions that have the identical sequence as that found in thegermline immunoglobulin gene from which the framework sequence derive,or the CDR sequences can be grafted onto framework regions that containone or more mutations as compared to the germline sequences. Forexample, it has been found that in certain instances it is beneficial tomutate residues within the framework regions to maintain or enhance theantigen-binding ability of the antibody (see e.g., U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).Frameworks that can be utilized as scaffolds on which to build theantibodies and antigen-binding fragments described herein include, butare not limited to VH1A, VH1B, VH3, Vk1, V12, and Vk2. Additionalframeworks are known in the art and may be found, for example, in thevBase data base on the world wide web atvbase.mrc-cpe.cam.ac.uk/index.php?&MMN position=1:1.

Accordingly, an embodiment of the present disclosure relates to isolatedβ-klotho binding antibodies, or antigen-binding fragments thereof,comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID Nos: 15, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions inthe framework region of such sequences, and further comprising a lightchain variable region having an amino acid sequence selected from thegroup consisting of SEQ ID Nos: 26 or 32, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions in the framework region of such sequences, wherein theheavy chain variable region does not comprise a heavy chain variableregion amino acid sequence set forth in Table 2, e.g., the amino acidsequence of SEQ ID NO: 43 or 55 and the light chain variable region doesnot comprise a light chain variable region amino acid sequence set forthin Table 2, e.g., the amino acid sequence of SEQ ID NO: 47 or 57.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Certain amino acid sequence motifs are known to undergopost-translational modification (PTM) such as glycosylation (i.e. NxS/T,x any but P), oxidation of free cysteines, deamidation (e.g. NG) orisomerization (e.g. DG). If present in the CDR regions, those motifs areideally removed by site-directed mutagenesis in order to increaseproduct homogeneity.

The process of affinity maturation is well described in the art. Amongmany display systems, phage display (Smith G P (1985) Science228:1315-1317) and display on eukaryotic cells such as yeast (Boder E Tand Wittrup K_(D) (1997) Nature Biotechnology 15: 553-557) seem to bethe most commonly applied systems to select for antibody-antigeninteraction. Advantages of those display systems are that they aresuitable for a wide range of antigens and that the selection stringencycan be easily adjusted. In phage display, scFv or Fab fragments can bedisplayed and in yeast display full-length IgG in addition. Thosecommonly applied methods allow selection of a desired antibody variantsfrom larger libraries with diversities of more than 10E7. Libraries withsmaller diversity, e.g. 10E3, may be screened by micro-expression andELISA.

Non-targeted or random antibody variant libraries can be generated forexample by error-prone PCR (Cadwell R C and Joyce G F (1994) MutagenicPCR. PCR Methods Appl. 3: S136-S140) and provide a very simple, butsometimes limited approach. Another strategy is the CDR directeddiversification of an antibody candidate. One or more positions in oneor more CDRs can be targeted specifically using for example degeneratedoligos (Thompson J et al. (1996) J. Mol. Biol. 256: 77-88) trinucloetidemutagenesis (TRIM) (Kayushin A L et al. (1996) Nucleic Acids Res. 24:3748-3755) or any other approach known to the art.

Accordingly, in another embodiment, the present disclosure providesisolated β-klotho-binding antibodies, or antigen-binding fragmentsthereof, consisting of a heavy chain variable region having a VH CDR1region consisting of an amino acid sequence selected from the grouphaving SEQ ID Nos: 6 and 9 or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID Nos: 6 and 9; a VH CDR2 region having an amino acidsequence of SEQ ID NO: 7 or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NO: 7; a VH CDR3 region having an amino acid sequenceof SEQ ID NO: 8, or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NO: 8; a VL CDR1 region having an amino acid sequence selectedfrom the group consisting of SEQ ID Nos: 19 or 31, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID Nos: 19 or 31; a VL CDR2region having an amino acid sequence of SEQ ID NO: 20, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NO: 20; and a VL CDR3region having an amino acid sequence of SEQ ID NO: 21, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NO: 21.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to β-klotho. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof, and include immunoglobulins of other animal species,preferably having humanized aspects. Single heavy-chain antibodies suchas those identified in camelids are of particular interest in thisregard. Novel frameworks, scaffolds and fragments continue to bediscovered and developed by those skilled in the art.

In one aspect, the present disclosure pertains to generatingnon-immunoglobulin based antibodies using non-immunoglobulin scaffoldsonto which CDRs of the present disclosure can be grafted. Known orfuture non-immunoglobulin frameworks and scaffolds may be employed, aslong as they comprise a binding region specific for the targetβ-klothoprotein. Known non-immunoglobulin frameworks or scaffoldsinclude, but are not limited to, fibronectin (Compound Therapeutics,Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich,Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, andAblynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., MountainView, CA), Protein A (Affibody AG, Sweden), and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

The fibronectin scaffolds are based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).The fibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). These fibronectin-based scaffolds are not an immunoglobulin,although the overall fold is closely related to that of the smallestfunctional antibody fragment, the variable region of the heavy chain,which comprises the entire antigen recognition unit in camel and llamaIgG. Because of this structure, the non-immunoglobulin antibody mimicsantigen-binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the present disclosure usingstandard cloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel α-helices and aβ-turn. Binding of the variable regions is mostly optimized by usingribosome display.

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 k_(D)a,compared to the molecular weight of antibodies, which is 150 k_(Dα). Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids. The proteinarchitecture is reminiscent of immunoglobulins, with hypervariable loopson top of a rigid framework. However, in contrast with antibodies ortheir recombinant fragments, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues, being justmarginally bigger than a single immunoglobulin domain. The set of fourloops, which makes up the binding pocket, shows pronounced structuralplasticity and tolerates a variety of side chains. The binding site canthus be reshaped in a proprietary process in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. One protein of lipocalin family, the bilin-binding protein(BBP) of Pieris Brassicae has been used to develop anticalins bymutagenizing the set of four loops. One example of a patent applicationdescribing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in W0200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions.

The present disclosure provides fully human antibodies that specificallybind to β-klotho protein. In certain aspects, compared to the chimericor humanized antibodies, the human β-klotho-binding antibodies of thepresent disclosure have further reduced antigenicity when administeredto human subjects.

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries ofcamelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present disclosure is a camelid antibodyor nanobody having specific affinity for β-klotho (e.g., humanβ-klotho). In certain embodiments herein, the camelid antibody ornanobody is naturally produced in the camelid animal, i.e., is producedby the camelid following immunization with β-klotho or a peptidefragment thereof, using techniques described herein for otherantibodies. Alternatively, the β-klotho-binding camelid nanobody isengineered, i.e., produced by selection for example from a library ofphage displaying appropriately mutagenized camelid nanobody proteinsusing panning procedures with β-klotho as a target as described in theexamples herein. Engineered nanobodies can further be customized bygenetic engineering to have a half life in a recipient subject of from45 minutes to two weeks. In a specific embodiment, the camelid antibodyor nanobody is obtained by grafting the CDRs sequences of the heavy orlight chain of the human antibodies of the present disclosure intonanobody or single domain antibody framework sequences, as described forexample in PCT/EP93/02214.

Bispecific Molecules and Multivalent Antibodies

In another aspect, the present disclosure features bispecific ormultispecific molecules comprising a β-klotho-binding antibody, or afragment thereof, of the present disclosure, for example, antibodyNOV005 or NOV006. An antibody of the present disclosure, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the present disclosure may in fact be derivatized orlinked to more than one other functional molecule to generatemulti-specific molecules that bind to more than two different bindingsites and/or target molecules; such multi-specific molecules are alsointended to be encompassed by the term “bispecific molecule” as usedherein. To create a bispecific molecule of the present disclosure, anantibody of the present disclosure can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other binding molecules, such as another antibody,antibody fragment, peptide or binding mimetic, such that a bispecificmolecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for β-klotho and asecond binding specificity for a second target epitope. For example, thesecond target epitope is another epitope of β-klotho different from thefirst target epitope.

Additionally, for the present disclosure in which the bispecificmolecule is multi-specific, the molecule can further include a thirdbinding specificity, in addition to the first and second target epitope.

In one aspect, the bispecific molecules of the present disclosurecomprise as a binding specificity at least one antibody, or an antibodyfragment thereof, including, e.g., a Fab, Fab′, F(ab′)2, Fv, or a singlechain Fv. The antibody may also be a light chain or heavy chain dimer,or any minimal fragment thereof such as a Fv or a single chain constructas described in Ladner et al. U.S. Pat. No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which VH and VL domainsare expressed on a single polypeptide chain, connected by a linker thatis too short to allow for pairing between the two domains on the samechain. The VH and VL domains pair with complementary domains of anotherchain, thereby creating two antigen-binding sites (see e.g., Holliger etal., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994Structure 2:1121-1123). Diabodies can be produced by expressing twopolypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VLconfiguration), or VLA-VHB and VLB-VHA (VL-VH configuration) within thesame cell. Most of them can be expressed in soluble form in bacteria.Single chain diabodies (scDb) are produced by connecting the twodiabody-forming polypeptide chains with linker of approximately 15 aminoacid residues (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology,2(1):21-36). scDb can be expressed in bacteria in soluble, activemonomeric form (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(34): 128-30; Wu et al., 1996 Immunotechnology,2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105;Ridgway et al., 1996 Protein Eng., 9(7):617-21). A diabody can be fusedto Fc to generate a “di-diabody” (see Lu et al., 2004 J. Biol. Chem.,279(4):2856-65).

Other antibodies which can be employed in the bispecific molecules ofthe present disclosure are murine, chimeric and humanized monoclonalantibodies.

Bispecific molecules can be prepared by conjugating the constituentbinding specificities, using methods known in the art. For example, eachbinding specificity of the bispecific molecule can be generatedseparately and then conjugated to one another. When the bindingspecificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No.78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)2 or ligand×Fab fusion protein. A bispecific molecule of thepresent disclosure can be a single chain molecule comprising one singlechain antibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

In another aspect, the present disclosure provides multivalent compoundscomprising at least two identical or different antigen-binding portionsof the antibodies of the present disclosure binding to β-klotho. Theantigen-binding portions can be linked together via protein fusion orcovalent or non covalent linkage. Alternatively, methods of linkage havebeen described for the bispecfic molecules. Tetravalent compounds can beobtained for example by cross-linking antibodies of the antibodies ofthe present disclosure with an antibody that binds to the constantregions of the antibodies of the present disclosure, for example the Fcor hinge region.

Trimerizing domain are described for example in Borean patent EP 1 012280B1. Pentamerizing modules are described for example inPCT/EP97/05897.

Antibodies with Extended Half Life

The present disclosure provides for antibodies (e.g., NOV005 or NOV006)that specifically bind to β-klotho protein which have an extendedhalf-life in vivo.

Many factors may affect a protein's half life in vivo. For examples,kidney filtration, metabolism in the liver, degradation by proteolyticenzymes (proteases), and immunogenic responses (e.g., proteinneutralization by antibodies and uptake by macrophages and dendriticcells). A variety of strategies can be used to extend the half life ofthe antibodies of the present disclosure. For example, by chemicallinkage to polyethyleneglycol (PEG), reCODE PEG, antibody scaffold,polysialic acid (PSA), hydroxyethyl starch (HES), albumin-bindingligands, and carbohydrate shields; by genetic fusion to proteins bindingto serum proteins, such as albumin, IgG, FcRn, and transferring; bycoupling (genetically or chemically) to other binding moieties that bindto serum proteins, such as nanobodies, Fabs, DARPins, avimers,affibodies, and anticalins; by genetic fusion to rPEG, albumin, domainof albumin, albumin-binding proteins, and Fc; or by incorporation intonanocarriers, slow release formulations, or medical devices.

To prolong the serum circulation of antibodies in vivo, inert polymermolecules such as high molecular weight PEG can be attached to theantibodies or a fragment thereof with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of the antibodies or via epsilon-amino groups present onlysine residues. To pegylate an antibody, the antibody, or fragmentthereof, typically is reacted with polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. The pegylation can be carried out by an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Linear or branched polymer derivatization that results in minimal lossof biological activity will be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or byion-exchange chromatography. PEG-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methodswell-known to those of skill in the art, for example, by immunoassaysdescribed herein. Methods for pegylating proteins are known in the artand can be applied to the antibodies of the present disclosure. See forexample, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawaet al.

Other modified pegylation technologies include reconstituting chemicallyorthogonal directed engineering technology (ReCODE PEG), whichincorporates chemically specified side chains into biosynthetic proteinsvia a reconstituted system that includes tRNA synthetase and tRNA. Thistechnology enables incorporation of more than 30 new amino acids intobiosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNAincorporates a nonnative amino acid any place an amber codon ispositioned, converting the amber from a stop codon to one that signalsincorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serumhalflife extension. This technology involves genetically fusing a300-600 amino acid unstructured protein tail to an existingpharmaceutical protein. Because the apparent molecular weight of such anunstructured protein chain is about 15-fold larger than its actualmolecular weight, the serum halflife of the protein is greatlyincreased. In contrast to traditional PEGylation, which requireschemical conjugation and repurification, the manufacturing process isgreatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymerpolysialic acid (PSA) to prolong the active life and improve thestability of therapeutic peptides and proteins. PSA is a polymer ofsialic acid (a sugar). When used for protein and therapeutic peptidedrug delivery, polysialic acid provides a protective microenvironment onconjugation. This increases the active life of the therapeutic proteinin the circulation and prevents it from being recognized by the immunesystem. The PSA polymer is naturally found in the human body. It wasadopted by certain bacteria which evolved over millions of years to coattheir walls with it. These naturally polysialylated bacteria were thenable, by virtue of molecular mimicry, to foil the body's defense system.PSA, nature's ultimate stealth technology, can be easily produced fromsuch bacteria in large quantities and with predetermined physicalcharacteristics. Bacterial PSA is completely non-immunogenic, even whencoupled to proteins, as it is chemically identical to PSA in the humanbody.

Another technology includes the use of hydroxyethyl starch (“HES”)derivatives linked to antibodies. HES is a modified natural polymerderived from waxy maize starch and can be metabolized by the body'senzymes. HES solutions are usually administered to substitute deficientblood volume and to improve the rheological properties of the blood.Hesylation of an antibody enables the prolongation of the circulationhalf-life by increasing the stability of the molecule, as well as byreducing renal clearance, resulting in an increased biological activity.By varying different parameters, such as the molecular weight of HES, awide range of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375.

Further, antibodies can be conjugated to albumin (e.g., human serumalbumin; HSA) in order to make the antibody or antibody fragment morestable in vivo or have a longer half life in vivo. The techniques arewell-known in the art, see, e.g., International Publication Nos. WO93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP413,622. In addition, in the context of a bispecific antibody asdescribed above, the specificities of the antibody can be designed suchthat one binding domain of the antibody binds to FGF21 while a secondbinding domain of the antibody binds to serum albumin, preferably HSA.

The strategies for increasing half life is especially useful innanobodies, fibronectin-based binders, and other antibodies or proteinsfor which increased in vivo half life is desired.

Antibody Conjugates

The present disclosure provides antibodies or fragments thereof thatspecifically bind to a β-klotho protein recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a heterologous protein or polypeptide (or fragmentthereof, preferably to a polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids) to generate fusionproteins. In particular, the present disclosure provides fusion proteinscomprising an antigen-binding fragment (e.g., a Fab fragment, Fdfragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VLdomain or a VL CDR) of an antibody described herein, for example, NOV005or NOV006, and a heterologous protein, polypeptide, or peptide. Methodsfor fusing or conjugating proteins, polypeptides, or peptides to anantibody or an antibody fragment are known in the art. See, e.g., U.S.Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and5,112,946; European Patent Nos. EP 307,434 and EP 367,166; InternationalPublication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991,Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J.Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA89:11337-11341.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the present disclosureor fragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to aβ-klotho protein may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide(SEQ ID NO: 64), such as the tag provided in a pQE vector (QIAGEN, Inc.,9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of whichare commercially available. As described in Gentz et al., 1989, Proc.Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine (SEQ IDNO: 64) provides for convenient purification of the fusion protein.Other peptide tags useful for purification include, but are not limitedto, the hemagglutinin (“HA”) tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984,Cell 37:767), and the “flag” tag.

In other embodiments, antibodies of the present disclosure or fragmentsthereof conjugated to a diagnostic or detectable agent. Such antibodiescan be useful for monitoring or prognosing the onset, development,progression and/or severity of a disease or disorder as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy. Such diagnosis and detection can accomplished bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidinlbiotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C),sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In),technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium(103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu,159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142 Pr,105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn,75Se, 113Sn, and 117Tin; and positron emitting metals using variouspositron emission tomographies, and noradioactive paramagnetic metalions.

The present disclosure further encompasses uses of antibodies orfragments thereof conjugated to a therapeutic moiety. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, an anti-angiogenicagent; or, a biological response modifier such as, for example, alymphokine.

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alph-emiters such as 213Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Methods of Producing Antibodies

Nucleic Acids Encoding the Antibodies

The present disclosure provides substantially purified nucleic acidmolecules which encode polypeptides comprising segments or domains ofthe β-klotho-binding antibody chains described above. Some of thenucleic acids of the present disclosure comprise the nucleotide sequenceencoding the heavy chain variable region shown in SEQ ID NO: 15, and/orthe nucleotide sequence encoding the light chain variable region shownin SEQ ID NO: 26 or 32. In a specific aspect, nucleic acid moleculesprovided herein are those identified in Table 1, for example, nucleicacid molecules comprising the sequence of SEQ ID NO: 16, 36, or 38encoding a VH, or nucleic acid molecules comprising the sequence of SEQID NO: 27, 54, 33 or 40 encoding a VL. Some other nucleic acid moleculesof the present disclosure comprise nucleotide sequences that aresubstantially identical (e.g., at least 65, 80%, 95%, or 99%) to thenucleotide sequences of those identified in Table 1, for example,nucleic acid molecules comprising the sequence of SEQ ID NO: 16, 36, or38 encoding a VH, or nucleic acid molecules comprising the sequence ofSEQ ID NO: 27, 54, 33 or 40 encoding a VL. When expressed fromappropriate expression vectors, polypeptides encoded by thesepolynucleotides are capable of exhibiting FGF21 antigen-bindingcapacity.

Also provided in the present disclosure are polynucleotides which encodeall or substantially all of the variable region sequence of a heavychain and/or a light chain of the β-klotho-binding antibody set forthherein, for example, those set forth in Table 1. In specific aspects,the present disclosure provides polynucleotides which encode all orsubstantially all of the VH and/or VL of a β-klotho-binding antibodyNOV005. In specific aspects, the present disclosure providespolynucleotides which encode all or substantially all of the heavy chainand/or light chain of a β-klotho-binding antibody NOV005. In specificaspects, the present disclosure provides polynucleotides which encodeall or substantially all of the VH and/or VL of a β-klotho-bindingantibody NOV006. In specific aspects, the present disclosure providespolynucleotides which encode all or substantially all of the heavy chainand/or light chain of a β-klotho-binding antibody NOV006.

Because of the degeneracy of the code, a variety of nucleic acidsequences will encode each of the immunoglobulin amino acid sequences.For example, SEQ ID Nos: 16 and 36 are two nucleic acid sequences whichencode for a VH of NOV005 and SEQ ID Nos: 27 and 54 are two nucleic acidsequences which encode for a VL of NOV005.

The nucleic acid molecules of the present disclosure can encode both avariable region and a constant region of the antibody. Some of nucleicacid sequences of the present disclosure comprise nucleotides encoding aheavy chain sequence that is substantially identical (e.g., at least80%, 90%, or 99%) to the heavy chain sequence set forth in SEQ ID NO:17. Some other nucleic acid sequences comprising nucleotide encoding alight chain sequence that is substantially identical (e.g., at least80%, 90%, or 99%) to the light chain sequence set forth in SEQ ID NO: 28or 34. In a specific aspects, nucleic acid molecules provided herein arethose identified in Table 1, for example, nucleic acid moleculescomprising the sequence of SEQ ID NO: 18, 37, 30, or 39 encoding a heavychain, or nucleic acid molecules comprising the sequence of SEQ ID NO:29, 51, 35, or 41 encoding a light chain.

In a specific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 16 encoding a VH. In a specificaspect, provided herein is a polynucleotide comprising the nucleic acidsequence of SEQ ID NO: 36 encoding a VH. In a specific aspect, providedherein is a polynucleotide comprising the nucleic acid sequence of SEQID NO: 38 encoding a VH. In a specific aspect, provided herein is apolynucleotide comprising the nucleic acid sequence of SEQ ID NO: 27encoding a VL. In a specific aspect, provided herein is a polynucleotidecomprising the nucleic acid sequence of SEQ ID NO: 54 encoding a VL. Ina specific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 29 encoding a VL. In a specificaspect, provided herein is a polynucleotide comprising the nucleic acidsequence of SEQ ID NO: 51 encoding a VL.

In a specific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 18 encoding a heavy chain. In aspecific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 37 encoding a heavy chain. In aspecific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 30 encoding a heavy chain. In aspecific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 39 encoding a heavy chain.

In a specific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 29 encoding a light chain. In aspecific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 51 encoding a light chain. In aspecific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 35 encoding a light chain. In aspecific aspect, provided herein is a polynucleotide comprising thenucleic acid sequence of SEQ ID NO: 41 encoding a light chain.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding a β-klotho-binding antibodyor its binding fragment. Direct chemical synthesis of nucleic acids canbe accomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Inniset al. (Ed.), Academic Press, San Diego, C A, 1990; Mattila et al.,Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the present disclosure are expression vectors and hostcells for producing the β-klotho-binding antibodies described herein,e.g., NOV005 or NOV006. Various expression vectors can be employed toexpress the polynucleotides encoding the β-klotho-binding antibodychains or binding fragments. Both viral-based and nonviral expressionvectors can be used to produce the antibodies in a mammalian host cell.Nonviral vectors and systems include plasmids, episomal vectors,typically with an expression cassette for expressing a protein or RNA,and human artificial chromosomes (see, e.g., Harrington et al., NatGenet 15:345, 1997). For example, nonviral vectors useful for expressionof the FGF21-binding polynucleotides and polypeptides in mammalian(e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A,B & C, (Invitrogen, San Diego, CA), MPSV vectors, and numerous othervectors known in the art for expressing other proteins. Useful viralvectors include vectors based on retroviruses, adenoviruses,adenoassociated viruses, herpes viruses, vectors based on SV40,papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors andSemliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev.Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding a β-klotho-bindingantibody chain or fragment. In some embodiments, an inducible promoteris employed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of a β-klotho-binding antibody chain or fragment.These elements typically include an ATG initiation codon and adjacentribosome binding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., Results Probl. CellDiffer. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedFGF21-binding antibody sequences. More often, the insertedβ-klotho-binding antibody sequences are linked to a signal sequencesbefore inclusion in the vector. Vectors to be used to receive sequencesencoding β-klotho-binding antibody light and heavy chain variabledomains sometimes also encode constant regions or parts thereof. Suchvectors allow expression of the variable regions as fusion proteins withthe constant regions thereby leading to production of intact antibodiesor fragments thereof. Typically, such constant regions are human.

The host cells for harboring and expressing the β-klotho-bindingantibody chains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present disclosure. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters will bepresent, such as the lactose promoter system, a tryptophan (trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences and the like, for initiating and completing transcription andtranslation. Other microbes, such as yeast, can also be employed toexpress FGF21-binding polypeptides of the present disclosure. Insectcells in combination with baculovirus vectors can also be used.

In some preferred embodiments, mammalian host cells are used to expressand produce the β-klotho-binding polypeptides of the present disclosure.For example, they can be either a hybridoma cell line expressingendogenous immunoglobulin genes (e.g., the 1D6.C9 myeloma hybridomaclone as described in the Examples) or a mammalian cell line harboringan exogenous expression vector (e.g., the SP2/0 myeloma cellsexemplified below). These include any normal mortal or normal orabnormal immortal animal or human cell. For example, a number ofsuitable host cell lines capable of secreting intact immunoglobulinshave been developed including the CHO cell lines, various Cos celllines, HeLa cells, myeloma cell lines, transformed B-cells andhybridomas. The use of mammalian tissue cell culture to expresspolypeptides is discussed generally in, e.g., Winnacker, FROM GENES TOCLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors formammalian host cells can include expression control sequences, such asan origin of replication, a promoter, and an enhancer (see, e.g., Queen,et al., Immunol. Rev. 89:49-68, 1986), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Theseexpression vectors usually contain promoters derived from mammaliangenes or from mammalian viruses. Suitable promoters may be constitutive,cell type-specific, stage-specific, and/or modulatable or regulatable.Useful promoters include, but are not limited to, the metallothioneinpromoter, the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts. (See generallySambrook, et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial vinons, fusionto the herpes virus structural protein VP22 (Elliot and O'Hare, Cell88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction.For long-term, high-yield production of recombinant proteins, stableexpression will often be desired. For example, cell lines which stablyexpress β-klotho-binding antibody chains or binding fragments can beprepared using expression vectors of the present disclosure whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells may be allowed to grow for 1-2 days in an enriched media beforethey are switched to selective media. The purpose of the selectablemarker is to confer resistance to selection, and its presence allowsgrowth of cells which successfully express the introduced sequences inselective media. Resistant, stably transfected cells can be proliferatedusing tissue culture techniques appropriate to the cell type.

Generation of Monoclonal Antibodies

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

Animal systems for preparing hybridomas include the murine, rat andrabbit systems. Hybridoma production in the mouse is a well establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present disclosure can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the present disclosure arehuman monoclonal antibodies. Such human monoclonal antibodies directedagainst β-klotho can be generated using transgenic or transchromosomicmice carrying parts of the human immune system rather than the mousesystem. These transgenic and transchromosomic mice include mice referredto herein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and K lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and K (chain loci (see e.g., Lonberg, etal., 1994 Nature 368(6474): 856-859). Accordingly, the mice exhibitreduced expression of mouse IgM or K, and in response to immunization,the introduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et al., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the present disclosure can beraised using a mouse that carries human immunoglobulin sequences ontransgenes and transchomosomes such as a mouse that carries a humanheavy chain transgene and a human light chain transchromosome. Suchmice, referred to herein as “KM mice”, are described in detail in PCTPublication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseβ-klotho-binding antibodies of the present disclosure. For example, analternative transgenic system referred to as the Xenomouse (Abgenix,Inc.) can be used. Such mice are described in, e.g., U.S. Pat. Nos.5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963 toKucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseβ-klotho-binding antibodies of the present disclosure. For example, micecarrying both a human heavy chain transchromosome and a human lightchain tranchromosome, referred to as “TC mice” can be used; such miceare described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise FGF21-bindingantibodies of the present disclosure.

Human monoclonal antibodies of the present disclosure can also beprepared using phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art or described in the examplesbelow. See for example: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S.Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the present disclosure can also beprepared using SCID mice into which human immune cells have beenreconstituted such that a human antibody response can be generated uponimmunization. Such mice are described in, for example, U.S. Pat. Nos.5,476,996 and 5,698,767 to Wilson et al.

Framework or Fc Engineering

Engineered antibodies of the present disclosure include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation may contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues can be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived. To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to thegermline sequence by, for example, site-directed mutagenesis. Such“backmutated” antibodies are also intended to be encompassed by thepresent disclosure.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell—epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the present disclosure may be engineered toinclude modifications within the Fc region, typically to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the presentdisclosure may be chemically modified (e.g., one or more chemicalmoieties can be attached to the antibody) or be modified to alter itsglycosylation, again to alter one or more functional properties of theantibody. Each of these embodiments is described in further detailbelow. The numbering of residues in the Fc region is that of the EUindex of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen”. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the present disclosure to thereby produce anantibody with altered glycosylation. For example, EP 1,176,195 by Hanget al. describes a cell line with a functionally disrupted FUT8 gene,which encodes a fucosyl transferase, such that antibodies expressed insuch a cell line exhibit hypofucosylation. PCT Publication WO 03/035835by Presta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods of Engineering Altered Antibodies

As discussed above, the β-klotho-binding antibodies having VH and VLsequences or full length heavy and light chain sequences shown hereincan be used to create new β-klotho-binding antibodies by modifying fulllength heavy chain and/or light chain sequences, VH and/or VL sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe present disclosure, the structural features of a β-klotho-bindingantibody of the present disclosure are used to create structurallyrelated β-klotho-binding antibodies that retain at least one functionalproperty of the antibodies of the present disclosure, such as binding tohuman β-klotho and also activating one or more functional properties ofthe FGF21-receptor complex (e.g., activating FGF21-receptor signaling).

For example, one or more CDR regions of the antibodies of the presentdisclosure, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, β-klotho-binding antibodies of the presentdisclosure, as discussed above. Other types of modifications includethose described in the previous section. The starting material for theengineering method is one or more of the VH and/or VL sequences providedherein, or one or more CDR regions thereof. To create the engineeredantibody, it is not necessary to actually prepare (i.e., express as aprotein) an antibody having one or more of the VH and/or VL sequencesprovided herein, or one or more CDR regions thereof. Rather, theinformation contained in the sequence(s) is used as the startingmaterial to create a “second generation” sequence(s) derived from theoriginal sequence(s) and then the “second generation” sequence(s) isprepared and expressed as a protein.

Accordingly, in another embodiment, the present disclosure provides amethod for preparing a 3-klotho-binding antibody (e.g., NOV005 orNOV006) comprising of a heavy chain variable region antibody sequencehaving a CDR1 sequence of SEQ ID NO: 6 or 9, a CDR2 sequence of SEQ IDNO: 7, and/or a CDR3 sequence of SEQ ID NO: 8; and a light chainvariable region antibody sequence having a CDR1 sequence of SEQ ID NO:19 or 31, a CDR2 sequence of SEQ ID NO: 20, and/or a CDR3 sequence ofSEQ ID NO: 21; optionally, altering at least one amino acid residuewithin the heavy chain variable region antibody sequence and/or thelight chain variable region antibody sequence to create at least onealtered antibody sequence; and expressing the altered antibody sequenceas a protein.

Accordingly, in another embodiment, the present disclosure provides amethod for preparing a 3-klotho-binding antibody optimized forexpression in a mammalian cell consisting of: a full length heavy chainantibody sequence comprising a sequence of SEQ ID NO: 17; and a fulllength light chain antibody sequence comprising a sequence of SEQ ID NO:28 or 34; optionally, altering at least one amino acid residue withinthe full length heavy chain antibody sequence and/or the full lengthlight chain antibody sequence to create at least one altered antibodysequence; and expressing the altered antibody sequence as a protein. Inone embodiment, the alteration of the heavy or light chain is in theframework region of the heavy or light chain.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences or minimal essential bindingdeterminants as described in US2005/0255552 and diversity on CDR1 andCDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the β-klotho-binding antibodies describedherein, which functional properties include, but are not limited to,specifically binding to human, cynomolgus, rat, and/or mouse β-klotho;and the antibody activates FGF21-mediated signaling, e.g.,FGF21-receptor-dependent signaling, in a FGFR1c_β-klotho_HEK293 pERKcell assay.

In certain embodiments of the methods of engineering antibodies of thepresent disclosure, mutations can be introduced randomly or selectivelyalong all or part of a β-klotho-binding antibody coding sequence and theresulting modified β-klotho-binding antibodies can be screened forbinding activity and/or other functional properties as described herein.Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies. In certain embodiments of the present disclosure antibodieshave been engineered to remove sites of deamidation. Deamidation isknown to cause structural and functional changes in a peptide orprotein. Deamindation can result in decreased bioactivity, as well asalterations in pharmacokinetics and antigenicity of the proteinpharmaceutical. (Anal Chem. 2005 Mar. 1; 77(5): 1432-9).

In certain embodiments of the present disclosure the antibodies havebeen engineered to increase pI and improve their drug-like properties.The p

of a protein is a key determinant of the overall biophysical propertiesof a molecule. Antibodies that have low pis have been known to be lesssoluble, less stable, and prone to aggregation. Further, thepurification of antibodies with low p

is challenging and can be problematic especially during scale-up forclinical use. Increasing the p

of the anti-β-klotho antibodies, or Fabs, of the present disclosureimproved their solubility, enabling the antibodies to be formulated athigher concentrations (>100 mg/ml). Formulation of the antibodies athigh concentrations (e.g. >100 mg/ml) offers the advantage of being ableto administer higher doses of the antibodies into eyes of patients viaintravitreal injections, which in turn may enable reduced dosingfrequency, a significant advantage for treatment of chronic diseasesincluding cardiovascular disorders. Higher pls may also increase theFcRn-mediated recycling of the IgG version of the antibody thus enablingthe drug to persist in the body for a longer duration, requiring fewerinjections. Finally, the overall stability of the antibodies issignificantly improved due to the higher pI resulting in longershelf-life and bioactivity in vivo. Preferably, the p

is greater than or equal to 8.2.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

Prophylactic and Therapeutic Uses

Antibodies that bind β-klotho as described herein (e.g., NOV005 orNOV006) and antigen-binding fragments thereof, can be used at atherapeutically useful concentration for the treatment of a disease ordisorder associated with aberrant FGF21 signaling (e.g., aberrantactivation of FGF21-mediated signaling and/or FGF21 receptor signaling),by administering to a subject in need thereof an effective amount of theantibodies or antigen-binding fragments of the present disclosure, e.g.,NOV005 or NOV006. The present disclosure provides a method of treatingFGF21-associated metabolic disorders by administering to a subject inneed thereof an effective amount of the antibodies of the presentdisclosure, e.g., NOV005 or NOV006. The present disclosure provides amethod of treating FGF21-associated cardiovascular disorders byadministering to a subject in need thereof an effective amount of theantibodies of the present disclosure, e.g., NOV005 or NOV006.

The antibodies of the present disclosure (e.g., NOV005 or NOV006) can beused, inter alia, to prevent treat, prevent, and improve FGF21associated conditions or disorders, including but not limited tometabolic, endocrine, and cardiovascular disorders, such as obesity,type 1 and type 2 diabetes mellitus, pancreatitis, dyslipidemia,nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), insulin resistance, hyperinsulinemia, glucose intolerance,hyperglycemia, hypertriglyceridemia, metabolic syndrome, acutemyocardial infarction, hypertension, cardiovascular disease,atherosclerosis, peripheral arterial disease, stroke, heart failure,coronary heart disease, kidney disease, diabetic complications,neuropathy, gastroparesis, disorders associated with severe inactivatingmutations in the insulin receptor, and other metabolic disorders, and inreducing the mortality and morbidity of critically ill patients.

The antibodies of the present disclosure (e.g., NOV005 or NOV006) can beused, inter alia, to treat, diagnose, ameliorate, improve, or prevent anumber of diseases, disorders, or conditions, including, but not limitedto metabolic diseases associated with insulin resistance, such as type 2diabetes mellitus, type 1 diabetes mellitus, insulin receptor mutationdisorders (INSR disorders, e.g., Type B insulin resistance),nonalcoholic fatty liver disease (NAFLD) and various forms of partiallipodystrophy including familial partial lipodystrophy and HIV-highlyactive antiretroviral therapy (HIV-HAART) induced partial lipodystrophyas well as diseases associated with insulin production (e.g., type 1diabetes mellitus), and in reducing the mortality and morbidity ofcritically ill patients.

Multiple inactivating mutations of the INSR have been described withvarying phenotypes. Patients typically present with severe resistance tothe action of insulin which advances to hyperglycemia at the time ofpuberty. The current standard of care is treatment with very high dosesof insulin when subjects become hyperglycemic, which typically isinadequate in controlling hyperglycemia.

In particular aspects, the antibodies of the present disclosure (e.g.,NOV005 or NOV006) can be used, inter altar, to treat or manage type 1diabetes mellitus, dyslipidemial, hyperglycemia, hypoglycemia, glucoseintolerance, hypertriglyceridemia, or HIV-HAART Induced PartialLipodystrophy.

The antibodies of the present disclosure can also be used in combinationwith other agents for the prevention, treatment, or improvement of FGF21associated disorders. For example, statin therapies may be used incombination with the FGF21 mimetic antibodies and antigen-bindingfragments of the present disclosure for the treatment of patients withcardiovascular or metabolic disorders.

In particular aspects, provided herein is a method of reducing bodyweight (e.g., by at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 12%, at least 15%, or atleast 20%) in a subject, comprising administering to a subject in needthereof an effective amount of a pharmaceutical composition comprisingan antibody or antigen-binding fragment described herein (e.g., NOV005or NOV006) which binds β-klotho and is capable of increasing theactivity β-klotho/FGFR1c receptor complex.

In particular aspects, provided herein is a method of reducing appetiteor food intake (e.g., by at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 15%,or at least 20%) in a subject, comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising an antibody or antigen-binding fragment described herein(e.g., NOV005 or NOV006) which binds β-klotho and is capable ofincreasing the activity of β-klotho/FGFR1c receptor complex.

In particular aspects, provided herein is a method of reducing (e.g., byat least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 12%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least 90%) plasma triglyceride (TG) concentrations orplasma total cholesterol (TC) concentrations in a subject, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment described herein (e.g., NOV005 or NOV006) which binds β-klothoand is capable of increasing the activity of β-klotho/FGFR1c receptorcomplex.

In specific aspects of the methods provided herein, the subject isafflicted with a metabolic disorder, such as obesity, type 1 and type 2diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholicsteatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucoseintolerance, hyperglycemia, hypertriglyceridemia, and metabolicsyndrome. In specific aspects of the methods provided herein, thesubject is afflicted with a cardiovascular disorder. In particularaspects, the subject is a human.

In certain aspects, provided herein is a method of treating or managingobesity in a subject, comprising administering to a subject in needthereof an effective amount of a pharmaceutical composition comprisingan antibody or antigen-binding fragment described herein (e.g., NOV005or NOV006) which binds β-klotho and is capable of increasing theactivity of β-klotho/FGFR1c receptor complex.

In certain aspects, provided herein is a method of treating or managingtype 2 diabetes in a subject, comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising an antibody or antigen-binding fragment described herein(e.g., NOV005 or NOV006) which binds β-klotho and is capable ofincreasing the activity of β-klotho/FGFR1c receptor complex.

In certain aspects, provided herein is a method of treating or managingtype 1 diabetes in a subject, comprising administering to a subject inneed thereof an effective amount of a pharmaceutical compositioncomprising an antibody or antigen-binding fragment described herein(e.g., NOV005 or NOV006) which binds β-klotho and is capable ofincreasing the activity of β-klotho/FGFR1c receptor complex.

In certain aspects, provided herein is a method of treating or managinglipodystropy, such as HIV-HAART induced partial lipodystrophy, in asubject, comprising administering to a subject in need thereof aneffective amount of a pharmaceutical composition comprising an antibodyor antigen-binding fragment described herein (e.g., NOV005 or NOV006)which binds β-klotho and is capable of increasing the activity ofβ-klotho/FGFR1c receptor complex.

In certain aspects, provided herein is a method of treating or managingNASH in a subject, comprising administering to a subject in need thereofan effective amount of a pharmaceutical composition comprising anantibody or antigen-binding fragment described herein (e.g., NOV005 orNOV006) which binds β-klotho and is capable of increasing the activityof β-klotho/FGFR1c receptor complex.

In certain aspects, provided herein is a method of treating or managingan insulin receptor mutation disorder in a subject, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment described herein (e.g., NOV005 or NOV006) which binds β-klothoand is capable of increasing the activity of β-klotho/FGFR1c receptorcomplex.

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions comprisingthe β-klotho-binding antibodies (intact or binding fragments) formulatedtogether with a pharmaceutically acceptable carrier. The compositionscan additionally contain one or more other therapeutic agents that aresuitable for treating or preventing, for example, cardiovasculardisorders. Pharmaceutically acceptable carriers enhance or stabilize thecomposition, or can be used to facilitate preparation of thecomposition. Pharmaceutically acceptable carriers include solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible.

A pharmaceutical composition of the present disclosure can beadministered by a variety of methods known in the art. The route and/ormode of administration vary depending upon the desired results. It ispreferred that administration be intravitreal, intravenous,intramuscular, intraperitoneal, or subcutaneous, or administeredproximal to the site of the target. The pharmaceutically acceptablecarrier should be suitable for intravitreal, intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion). Depending on the route of administration, theactive compound, i.e., antibody, bispecific and multispecific molecule,may be coated in a material to protect the compound from the action ofacids and other natural conditions that may inactivate the compound. Ina specific aspect, a pharmaceutical composition comprising aβ-klotho-binding antibody described herein, such as antibody NOV005 orNOV006, for use in the methods provided herein, is administeredsubcutaneously. In a specific aspect, a pharmaceutical compositioncomprising a β-klotho-binding antibody described herein, such asantibody NOV005 or NOV006, for use in the methods provided herein, isadministered intravenously.

The composition should be sterile and fluid. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

Pharmaceutical compositions of the present disclosure can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20th ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. Typically, a therapeutically effective dose orefficacious dose of the β-klotho-binding antibody is employed in thepharmaceutical compositions of the present disclosure. Theβ-klotho-binding antibodies are formulated into pharmaceuticallyacceptable dosage forms by conventional methods known to those of skillin the art. Dosage regimens are adjusted to provide the optimum desiredresponse (e.g., a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of factors including the activity ofthe particular compositions of the present disclosure employed, or theester, salt or amide thereof, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors.

A physician or veterinarian can start doses of the antibodies of thepresent disclosure employed in the pharmaceutical composition at levelslower than that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, effective doses of the compositions of the present disclosure,for the treatment of a cardiovascular disorders described herein varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. In a particularembodiment, for systemic administration with an antibody, the dosageranges from about 0.0001 to 100 mg/kg, or from 0.01 to 15 mg/kg, of thehost body weight. An exemplary treatment regime entails systemicadministration once per every two weeks or once a month or once every 3to 6 months. An exemplary treatment regime entails systemicadministration once per every two weeks or once a month or once every 3to 6 months, or as needed (PRN).

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels ofβ-klotho-binding antibody in the patient. In addition alternative dosingintervals can be determined by a physician and administered monthly oras necessary to be efficacious. In some methods of systemicadministration, dosage is adjusted to achieve a plasma antibodyconcentration of 1-1000 μg/ml and in some methods 25-500 μg/ml.Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, humanized antibodies show longer half life thanthat of chimeric antibodies and nonhuman antibodies. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example 1: Antibody Screening and Production Preparation of Human FGFR1cβ-Klotho 300.19 Cells for Use as an Antigen

300.19 cells which stably expressed the human FGFR1c (1-386 aa) andβ-klotho were generated for use as a whole cell antigen. The full-lengthcDNA encoding human β-klotho (GenBank Accession number NM_175737) wascloned into the EcoRI and EcoRV sites of pEF1/Myc-His B (Invitrogen Cat.#V92120). The cDNA encoding amino acids 1-386 of human FGFR1c (GenBankAccession number NM_023106) was cloned into the BamHI and NotI sites ofpEF6/Myc-His B (Invitrogen, Cat. number V96220). In both constructs aKozak sequence (CACC) was included immediately before the start codonand a stop codon was added before the Myc-His tag in the vector. Murinepre-B 300-19 cells were co-transfected with β-klotho and FGFR1c plasmidsby electroporation using the Amaxa Nucleofector device and Nucleofectorkit (Lonza, Cat #VCA-1003). Stable clones were selected using 1 mg/mlGeneticin (Invitrogen, Cat #10131) and 8 μg/m1Blasticidin (Invitrogen,Cat #46-1120) for 3 weeks.

Preparation of FGFR/β-Klotho-Expressing HEK293 Cells for Use in CellAssays

To test the binding specificity, functional activity, or orthologcross-reactivity of β-klotho antibodies, HEK293 cells stably expressinghuman FGFR1c β-klotho, human GFR2c_β-klotho, human FGFR3 c_β-klotho,human FGFR4_β-klotho, or cynomolgus monkey FGFR1c_β-klotho weregenerated using standard Lipofectamine 2000 transfection and cell cloneselection methods.

The following mammalian expression plasmids encoding full-length humanβ-klotho (NM_175737), human FGFR1c (NM 023106), human FGFR2c(NP_001138387), human FGFR3c (NP_000133), or human FGFR4 (NP_998812)cDNAs were used: for cynomolgus monkey β-klotho, the full-lengthsequence was PCR amplified from cynomolgus monkey adipose tissue cDNA(BioChain, Cat. #CJ 534003-Cy) with primers based on the human andrhesus monkey β-klotho sequences, and cloned. The cynomolgus monkeyFGFR1c cDNA was cloned from cynomolgus monkey adipose tissue cDNA(BioChain, Cat. #C1534003-Cy) using primers based on the human FGFR1csequence (#NM 023106) and was shown to be 100% identical at the aminoacid level to human FGFR1c. Hence, the human FGFR1c cDNA constructdescribed above was used to make HEK293 cells which stably expressedcynomolgus monkey β-klotho and human FGFR1c (#NM 023106) since the humanand cynomolgus monkey FGFR1c amino acid sequences are identical.

Determining the Binding Affinity of Antibodies to β-Klotho

Binding affinities of antibodies were determined using Biacore kineticanalysis. A Series S Sensor Chip CMS (GE Healthcare, Cat. No.BR-1005-30) was equilibrated to ambient temperature for approximately 30minutes The system was primed with 1×HBS-EP+buffer (10×soln, GEHealthcare, Cat. No. BR-1006-69) and the chip was loaded into theBiacore instrument. The chip was normalized using BIAnormalizing (GEHealthcare, Cat. No. BR-1006-51) solution and the chip was conditionedwith 50 mM NaOH using a flow rate of 60 μL/minute for all flow paths.The 30 second injection was repeated three times with a wait time of 60seconds. 10 mM sodium acetate pH 5.0 was injected at 60 μL/minute for120 seconds and repeated twice. After a new cycle was started forimmobilizing the anti-human IgG Fc using the Human Antibody Capture Kit(GE Healthcare, Cat. No. BR-1008-39). The anti-human Fc Ab was dilutedto 50 μg/mL in immobilization buffer (10 mM sodium acetate pH 5.0). Theamine coupling reagents were mixed 1:1 (EDC and NHS) and injected for 7minutes at 10 μL/minute. Then anti-human Fc Ab was injected for 6minutes at 10 L/minute. Lastly ethanolamine was injected for 7 minutesat 10 μL/minute with a wait time of 60 seconds. This should result inimmobilization of approximately 8000-10000 RU. Then we performed testinjections for an Rmax of 10 RUs (capture levels=˜28 RUs) for NOV004,NOV005 and NOV006. This was done using a flow rate of 10 uL/mL and a 3 MMgCl2 regeneration buffer. Each flow cell was evaluated each time due tochanges in chip surface, affinity of IgGs, IgG protein quality anddifferences in dilutions. We started with an IgG concentration of 0.5ug/mL and increased (or decreased) the concentration depending on theresults from the sample injections. For example, if 100 RU was observedin 15 seconds, then we diluted the Ab solution to 0.25 ug/mL andrepeated the injections. After capturing parameters were determined foreach antibody in each flow cell, human β-klotho at variousconcentrations was passed over the chip and ka (1/Ms), kd (1/s), K_(D)(M), and Rmax (RU) were calculated by Biacore kinetics.

Determining if Different Antibodies Bind Competitively to β-Klotho

Forte Bio was used to determine if antibodies competitively bound tohuman β-klotho. All samples and reagents were diluted in 10× kineticsbuffer (Forte Bio cat #18-1092) with PBS buffer with a 1/10 (v/v) ratio.Human β-klotho (R and D Systems 5889-KB-050) was diluted with kineticbuffer to the desired concentration (5 ug/ml). Human β-klotho was loadedonto an anti-his sensor (Forte Bio, cat #18-5114) for 20 seconds, thenantibody 1 was loaded for 400 seconds onto the sensor until saturationconditions were reached (200 nM). Lastly the competing antibody wasloaded onto the sensor for 100 seconds at 200 nM in the presence of 200nM antibody 1. The absence of a second binding signal indicates that theantibodies compete for binding to human β-klotho.

Measuring FGFR β-Klotho Receptor Activation Using a pERK Cell Assay

Standard techniques were used for cell culture and to measurephospho-ERK 1/2 (pERK) levels. Briefly, HEK293 cells stably expressinghuman FGFR1c_β-klotho, human FGFR2c_β-klotho, human FGFR3c_β-klotho,human FGFR4_β-klotho, or cynomolgus monkey FGFR1c_β-klotho weremaintained in DMEM medium (Invitrogen, 11995) containing 10% FBS(Hyclone, SH30071), blasticidin (Invitrogen, A1113902), and Geneticin(Invitrogen, 10131035) at 37° C. in 5% CO₂. Cells were plated into384-well poly-D-lysine-coated plates (BD Biosciences, 354663) andincubated overnight at 37° C. in 5% CO₂, followed by serum-starvation.

Hybridoma supernatants or β-klotho antibodies were diluted in Freestyle293 media and various concentrations of the antibodies were added to theplate. Following incubation, the cells were washed, then lysed withlysis buffer. Cell lysates were transferred to a 384-well assay plate(PerkinElmer, Cat. #6008280) and the AlphaScreen SureFire™ pERK 1/2 Kit(Perkin Elmer, TGRES10K) was used to measure phospho-ERK 1/2 levels.Plates were read on the EnVision 2104 multi-label reader (Perkin Elmer)using standard AlphaScreen settings. Dose-response data was graphed aspERK activity fold over basal versus protein concentration to determineEC50 values using the equation Y=Bottom+(Top-Bottom)/(1+10{circumflexover ( )}((Log EC₅₀-X)×HillSlope)) and GraphPad Prism 5 Software.

Preparation of Monoclonal Antibodies

Anti-human_β-klotho antibodies were generated in Balb/c (JacksonLaboratory strain: BALB/cJ) or Bcl2 22 wehi (Jackson Laboratory strain:C.Cg-Tg(BCL2)22Wehi/J) mice by whole cell immunizations essentially asdescribed in Dreyer et al. (2010) (Dreyer A M et. al. (2010) BMCBiotechnology 10:87).

Briefly, 1×10⁷ human FGFR1c_β-klotho_300.19 cells were injected intoBalb/c mice six times at 10 to 30 day intervals. The first whole cellinjections were done with Freund's Complete Adjuvant (Sigma-AldrichF5881). Cells and adjuvant were not mixed, but injected separately intwo close, but distinct subcutaneous sites. These were followed later byintraperitoneal injections of the same cells with either Sigma AdjuvantSystem (Sigma-Aldrich S6322) or without adjuvant.

Using Bcl2 22 wehi mice, 1×10⁷ human FGFR1c_β-klotho_300.19 cells wereinjected into these animals four times at seven day intervals. The firstinjections were done with Freund's Complete Adjuvant (Sigma-AldrichF5881). Cells and adjuvant were injected separately in two sets of twoclose, but distinct subcutaneous sites Subsequent injections of cellswere done subcutaneously without adjuvant.

Immune responses in the immunized mice were measured by afluorescence-activated cell sorting (FACS) assay. Serum from theimmunized mice diluted 1,000- or 10,000-fold was used to stain humanFGFR1c_β-klotho_HEK and human FGFR1c_β-klotho_300.19 cells, followed byan allophycocyanin (APC) secondary anti-murine IgG detection antibody(Jackson ImmunoResearch Cat #115-136-071). Fluorescence was read on aBecton Dickinson LSRII or Foressa flow cytometer. Four mice with thehighest titer were chosen for electrofusions.

Hybridoma Screening, Subcloning, and Selection

2×10⁸ spleenoctyes and 5×10⁷ fusion partner FO cells (ATCC, CRL-1646)were washed in Cytofusion Medium (LCM-C, Cyto Pulse Sciences) and fusedusing a Hybrimune Waveform Generator (Cyto Pulse Sciences, modelCEEF-50B) according to manufacturer's specification with a peak pulse of600 volts. Cells were plated into 384 well plates at a calculateddensity of 3,000 FO cells per well and cultured in HAT selection media(Sigma-Aldrich Cat. H0262).

The primary screen was performed using a high throughput FACS platform(Anderson, Paul. Automated Hybridoma Screening, Expansion, Archiving andAntibody Purification. In: 3rd Annual 2014 SLAS Conference. Jan. 18-22,2014, San Diego, CA). Briefly, hybridoma supernatants were incubatedwith human FGFR1c_β-klotho stably expressing and non-expressing celllines and antibody binding was determined with an anti-murine IgG-APCsecondary antibody (Jackson ImmunoReseach Cat #115-136-071).

Antibodies from each hybridoma supernatant were tested for bindingsimultaneously against four barcoded cell lines: 300.19 parental cells,human FGFR1c_β-klotho 300.19 cells, parental HEK 293 cells, and humanFGFR1c_β-klotho HEK 293 cells. 348 hits were chosen in the primaryscreen. Primary hits were expanded in 96-well plates and binding wasconfirmed again on human FGFR1c_β-klotho HEK 293 cells by FACS, yielding122 confirmed hits. HAT (hypoxanthine-aminopterin-thymidine)media-containing supernatants of 115 FACS binding reconfirmed hits wereprofiled for cell activation of the human FGFR1c_β-klotho receptorcomplex using the phospho-ERK 1/2 assay described herein.

Hybridomas with the highest phospho-ERK 1/2 cell activity in theresupernatants were expanded and IgGs were purified from theirsupernatants. Purified IgGs from 74 hybridomas were profiled for cellactivation of the human FGFR1c_β-klotho receptor complex using thephospho-ERK 1/2 assay described in Example 2. IgGs from hybridomas withthe best potency for phospho-ERK 1/2 activation of the humanFGFR1c_β-klotho receptor complex were profiled for orthologcross-reactivity to the cynomolgus monkey FGFR1c β-klotho receptorcomplex and selectivity for the human FGFR2c)_β-klotho and humanFGFR3c_β-klotho receptor complexes using the phospho-ERK 1/2 assaydescribed in Example 2. On the basis of these profiling results, a fewhybridoma clones, e.g., 127F19, were selected for further profiling. Inparticular, the most potent, purified IgGs, such as clone 127F19, wereprofiled for cross-reactivity and shown to activate cynomolgus monkeyFGFR1c_β-klotho; but not human FGFR2_β-Klotho, FGFR3c_β-Klotho,FGFR4_β-Klotho or Klotho FGFR. The selected IgGs, for example clone127F19, bound to β-Klotho or FGFR1c_β-Klotho expressing cells, and notFGFR1c alone expressing cells.

To evaluate 127F19 signalling in cells expressing α-klotho, HEK293 cellswere transfected with α-klotho, Egrl-luciferase and Renilla luciferase.Briefly, HEK293 cells were cultured in DMEM, 10% FBS and plated at 30000cells/well and transfected with Klotho, Egr-1-luc and TK-Rennila usingLipofectamine 2000. Next day, FGF23, FGF21, and 127F19 were diluted tothe indicated concentration in DMEM supplemented with 0.1% FBS and addedto transfected cells overnight. Luciferase activities were detected byDual-Glo luciferase assay kit (Promega, E2920) according tomanufacturer's instruction. As expected, FGF23, which requires α-klothoexpression for its signaling, showed strong luciferase expression.However, neither FGF21 or 127F19 showed any significant luciferaseexpression, suggesting that α-klotho does not act as co-receptor forFGF21 or these FGF21 mimetic antibodies.

Humanization and Affinity-Maturation of Monoclonal AntibodiesHumanization

The process of humanization is well described in the art (Jones P T etal. (1986) Nature 321: 522-525; Queen C et al. (1989) PNAS USA 86:10029-10033; Riechmann L et al. (1988) Nature 33:323-327; Verhoeyen Metal. (1988) Science 239: 1534-1536). The term humanization describes thetransfer of the antigen-binding site of a non-human antibody, e.g. amurine derived antibody, to a human acceptor framework, e.g. a humangermline sequence (Retter I et al. (2005). Nucleic Acids Res.33:D671-D674).

The main rationale for humanizing an antibody is seen in minimizing therisk of developing an immunogenic response to the antibody in humans(Rebello P R et al. (1999) Transplantation 68: 1417-1420). Theantigen-binding site comprises the complementary determining regions(CDRs) (Chothia C and Lesk A M (1987) Journal of Molecular Biology 196:901-917; Kabat E et al. (1991) Anon. 5th Edition ed; NIH Publication No.91: 3242) and positions outside the CDR, i.e. in the framework region ofthe variable domains (VL and VH) that directly or indirectly affectbinding. Framework residues that may directly affect binding can, forexample, be found in the so called “outer” loop region located betweenCDR2 and CDR3. Residues that indirectly affect binding are for examplefound at so called Vernier Zones (Foote J, Winter G. (1992) Journal ofMolecular Biology 224:4 87-499). They are thought to support CDRconformation. Those positions outside the CDRs are taken into accountwhen choosing a suitable acceptor framework to minimize the number ofdeviations of the final humanized antibody to the human germlineacceptor sequence in the framework regions.

Multiple human germline acceptor frameworks were tested for humanizationof both light chain and heavy chain. For example, human frameworksVBase_VH4_4-30.1 and VBase_VH3_3-21 were tested for humanization of theheavy chain, and human frameworks VBase_VK1_O18 and Vbase_VK3_L25 weretested for humanization of the light chain.

Sequence Optimization Affinity Maturation

Certain amino acid sequence motifs are known to undergopost-translational modification (PTM) such as glycosylation (i.e. NxS/T,x any but P), oxidation of free cysteines, deamidation (e.g. NG) orisomerization (e.g. DG). If present in the CDR regions, those motifs areideally removed by site-directed mutagenesis in order to increaseproduct homogeneity.

The process of affinity maturation is well described in the art. Amongmany display systems, phage display (Smith G P, 1985, Filamentous fusionphage: novel expression vectors that display cloned antigens on thevirion surface. Science 228:1315-1317) and display on eukaryotic cellssuch as yeast (Boder E T and Wittrup K_(D), 1997, Yeast surface displayfor screening combinatorial polypeptide libraries. Nature Biotechnology15: 553-557) seem to be the most commonly applied systems to select forantibody-antigen interaction. Advantages of those display systems arethat they are suitable for a wide range of antigens and that theselection stringency can be easily adjusted. In phage display, scFv orFab fragments can be displayed and in yeast display full-length IgG inaddition. Those commonly applied methods allow selection of a desiredantibody variants from larger libraries with diversities of more than10E7. Libraries with smaller diversity, e.g. 10E3, may be screen bymicro-expression and ELISA. Non-targeted or random antibody variantlibraries can be generated for example by error-prone PCR (Cadwell R Cand Joyce G F, 1994, Mutagenic PCR. PCR Methods Appl. 3: S136-S140) andprovide a very simple, but sometimes limited approach. Another strategyis the CDR directed diversification of an antibody candidate. One ormore positions in one or more CDRs can be targeted specifically usingfor example degenerated oligos (Thompson J et al., 1996, Affinitymaturation of a high-affinity human monoclonal antibody against thethird hypervariable loop of human immunodeficiency virus: use of phagedisplay to improve affinity and broaden strain reactivity. J. Mol. Biol.256: 77-88) trinucloetide mutagenesis (TRIM) (Kayushin A L et al., 1996,A convenient approach to the synthesis of trinucleotidephosphoramidites—synthons for the generation of oligonucleotide/peptidelibraries. Nucleic Acids Res. 24: 3748-3755) or any other approach knownto the art. Amino acid modifications were made to humanizationcandidates to remove PTM.

Generation of Expression Plasmids

DNA sequences coding for humanized VL and VH domains were ordered atGeneArt™ (Life Technologies Inc. Regensburg, Germany) including codonoptimization for homosapiens. Sequences coding for VL and VH domainswere subcloned by cut and paste from the GeneArt derived vectors intoexpression vectors suitable for secretion in mammalian cells. The heavyand light chains were cloned into individual expression vectors to allowco-transfection. Elements of the expression vector include a promoter(Cytomegalovirus (CMV) enhancer-promoter), a signal sequence tofacilitate secretion, a polyadenylation signal and transcriptionterminator (Bovine Growth Hormone (BGH) gene), an element allowingepisomal replication and replication in prokaryotes (e.g. SV40 originand ColE1 or others known in the art) and elements to allow selection(ampicillin resistance gene and zeocin marker).

Expression and Purification of Humanized Antibody Candidates

Human Embryonic Kidney cells constitutively expressing the SV40 large Tantigen (HEK293-T ATCC11268) are one of the commonly used host celllines for transient expression of humanized and/or optimized IgGproteins. Transfections were performed using PEI (Polyethylenimine, MW25.000 linear, Polysciences, USA Cat. No. 23966) as transfectionreagent.

A first purification was performed by affinity on a HiTrap ProtAMabSelect®SuRe column. The eluate was tested for aggregation (SEC-MALS)and purity (SDS-PAGE, LAL and MS). If needed, pools from the firstpurification were loaded a SPX (Hi Load 16/60 Superdex 200 grade 120 mL(GE-Helthcare). NOV004, NOV005, and NOV006 are humanized mAbs derivedfrom the mouse hybridoma 127F19. The IgG1 L234A/L235A (LALA) or IgG1KD265A/P329A (DAPA) isotypes were selected as preventative measures toreduce the antibody's ability to promote antibody-dependentcell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC) (see, e.g., Hezareh M et al. (2001) Journal of Virology 75:12161-12168). Humanization candidates NOV004, NOV005, and NOV006, whichare IgG1 (DAPA) isotypes, were expressed and purified as described.

Example 2: In Vitro Charactionization of Monoclonal Antibodies

In vitro work was done to show the binding and cell activity propertiesof NOV004, NOV005, and NOV006. Biacore as described in Example 1 wasused to estimate the K_(D)s of the mAbs to human β-klotho. NOV004,NOV005, and NOV006 have K_(D)s calculated to be about 3×E-10M, 3×E-10M,and 4×E-10M, respectively.

The pERK assay as described in Example 1 was used to profile mAbs forFGFR_β-klotho receptor activity. NOV004 activated the human andcynomolgus monkey FGFR1c_β-klotho receptor complex with ECsos calculatedto be about 3 nM and 20 nM, respectively (FIG. 1 ). NOV005 activated thehuman and cynomolgus monkey FGFR1c_β-klotho receptor complex with anEC_(5os) calculated to be about 3 nM and 16 nM, respectively (FIG. 1 ).NOV006 activated the human and cynomolgus monkey FGFR1c β-klothoreceptor complex with an EC_(5os) calculated to be about 4 nM and 18 nM,respectively (FIG. 1 ). NOV005, NOV006, and NOV006 did not activatehuman FGFR2c_β-klotho, FGFR3c_β-klotho, or FGFR4 j-klotho receptorcomplexes (FIGS. 2A-2C). The mAbs were profiled for FGF23 activity asdescribed in Example 1. NOV005, NOV006, and NOV004 did not exhibit FGF23activity (FIG. 3 ). Forte Bio data shows that NOV005 and NOV006 competewith NOV004 for binding to human β-klotho (FIG. 4 ).

Epitope mapping studies by hydrogen deuterium exchange of human β-klothoextracellular domain with a version of NOV004 that has a human IgG1-LALAisotype show that the following peptides are significantly protected inthe mAb-β-klotho ECD complex: 246-265, 343-349, 421-429, 488-498,509-524, 536-550, 568-576, 646-669, 773-804, 834-857, and 959-986 aa;and that the following regions are most strongly protected: 246-265,536-550, 834-857 and 959-986 aa (data not shown; see PCT InternationalApplication Publication No. WO 2017/021893, which is hereby incorporatedby reference in its entirety). These studies, in combination with thebinding, activity and competition data, indicate that NOV005 and NOV006would also significantly protect such β-klotho ECD regions.

Example 3: Pharmacokinetic Profiles of Monoclonal Antibodies in RatAnimals and Maintenance Conditions

Animal care and husbandry were provided according to the Guide for theCare and Use of Laboratory Animals (Institute of Laboratory AnimalResources, National Research Council). All procedures were governed bythe standards set forth by the US Department of Health and HumanServices and performed according to protocol approved by the NovartisInstitutes for BioMedical Research (NIBR) Animal Care and Use Committee.Male, Sprague-Dawley rats (n=3/group) were housed in solid-bottom cageson a rack equipped to automatically provide water ad libitum, maintainedon a 12 hr light/dark cycle (6 am to 6 pm), and given free access tostandard rodent chow (Harlan-Teklad; Frederick, MD; cat #8604). Thevivarium was maintained between 68 and 76° F. with 30 to 70% humidity.

NOV004, NOV005, or NOV006 Preparation and Dosing

Stock solutions of NOV004, NOV005, and NOV006 in 10 mM Hi s/Hi s-HCl,220 mM sucrose were thawed under refrigeration prior to use. On themorning of dosing, all three antibodies were diluted to approximately 3mg/mL in PBS and appropriate volumes were drawn into dosing syringes (3mL/kg) and kept at room temperature until administration. Animals wereplaced in tube restrainers and administered either NOV004, NOV005, orNOV006 via intravenous (IV) injection into the tail vein (10 mg/kg).

Blood Sample Collection

Blood samples were collected on day −3 (Baseline), day 0 (1 and 6 hpost-dose), and days 1, 2, 3, 4, 8, 16 and 28 post-dose. All time pointswere timed from the end of administration of the dose given on day 0. Ateach timepoint, approximately 70 μl (70 μL) of blood was collected intoBD Microtainer collection/separator tubes with EDTA (Becton, Dickinson,and Company; Franklin Lakes, NJ; cat #365973). Pressure was applied withgauze to stop the bleeding. Samples were centrifuged for 10 min at20,817×g, and then ˜30 μL plasma was transferred to 0.2 mL Thermo-striptube (Thermo-Scientific; Pittsburgh, PA; cat #AB-0451) and frozen at 80°C. Rats were returned to their home cage after each collection.

Measurement of Plasma Total NOV004, NOV005, or NOV006 Concentrations

Human IgG (i.e. NOV004, NOV005, or NOV006) in rat plasma was quantifiedusing a custom sandwich immunoassay with a Goat anti-Human Fc-gammaantibody (KPL #109,005-098) as capture antibody and a goatanti-human-IgG with an HRP label as detection antibody. The captureantibody (2 μg/mL in PBS, 30 μL/well) was coated on 384-well, white,microtiter plates (Greiner Bio-One; Monroe, NC; cat no. 781074). Theplates were incubated overnight at room temperature (RT) withoutshaking. After aspirating the coating solution without washing, 90 μL of1×Milk Diluent/Blocking solution (KPL; Gaithersburg, MD; cat no.50-82-01) was added to each well and the plates were incubated for 2 hat RT. At the end of the incubation, the solution was aspirated and theplates were stored in foil pouches with desiccant at −80° C.

On the day of the assay, sixteen NOV004, NOV005, and NOV006 standardconcentrations, ranging from 0.244-4000 pM, were prepared by serialdilution in Casein buffer, including a buffer negative control. Allstudy samples were diluted 1:50 manually in Casein buffer and thenserially diluted 5-fold using a Biomek Fx for a total of threedilutions. The plates were incubated for 2 h at RT and then washed 3times with phosphate wash buffer (90 μL/well). HRP-labeled goatanti-human-IgG (400 ng/mL in Casein buffer, 30 μL/well) was added toeach plate and the plates were incubated for 1 h at RT. The plates werewashed 3 times with phosphate wash buffer (90 μL/well), and then KPLLumiGLO Chemiluminescent Substrate was added (30 μL/well; cat no.54-61-00). Chemiluminescence was read immediately on a SpectraMax M5plate reader (Molecular Devices) at all wavelengths with 50 msintegration time. Human Fc concentrations (pM) in plasma samples wereinterpolated from the NOV004, NOV005, or NOV006 standard curves,multiplied by dilution factors, and converted to nM concentrations.

Animals exhibited mean C_(m) of approximately 200 μg/mL at 1 h after IVadministration of NOV004, NOV005, or NOV006. NOV004, NOV005, and NOV006exhibited equivalent PK profiles in Sprague-Dawley rats (FIGS. 5A-5C).

Example 4: Developability and Formulation Assessments

Production process and formulation studies for antibodies NOV004,NOV005, and NOV006 were carried out. The observed formation of highmolecular weight species (HMW) during previous assessments of NOV004suggested a high degree of aggregation in solution and prompted thedevelopment and assessment of other FGF21 mimetic antibodies which havecomparable functional activities but better production and formulationprofiles, e.g., none to little (e.g., less than 20%, less than 15%, lessthan 10%, or less than 5%) observed formation of HIVIW.

DNA sequences coding for antibodies NOV005 and NOV006, including codonoptimization for homosapiens, were ordered at GeneArt™ (LifeTechnologies Inc. Regensburg, Germany) and ATUM (Menlo Park, CA).Vectors expressing NOV004, NOV005 and NOV006 were linearized andtransfected into a CHO cell line. Pools were selected using 10 nMmethotrexate (MTX) until recovered to >95% viability. One pool for eachmolecule was selected for wave production and scaled up appropriately.Culture supernatant from the wave production were harvested after 13days and filtered.

The wave harvest material of NOV005 and NOV006 was processed using twocolumn chromatography purification process for standardantibodies—captured using affinity chromatography (resin—GE HealthcareMabSelect SuRe) and polish using cation exchange chromatography (CEC)(resin—Fractogel EMD SO3—). The captured material was subject to ViralInactivation (adjustment of pH to 3.5, incubation at this pH at roomtemperature for 70 min followed by adjustment of pH to 5.0) and sterilefiltration before processing through CEC. After CEC, the material wasdiafiltered to buffer exchange into 10 mM Histidine/Histidine-HCl, pH5.0 using tangential flow filtration. Subsequently, the material wasconcentrated to about 200 mg/mL to provide material for formulationstudies. For example, the antibodies were assessed in formulationbuffers, and certain parameters were determined, in particular,formation of HMW after 4 weeks at 40° C. Table 3 below summarizes datafrom an exemplary experiment assessing the formation of HMW observed insamples of NOV004, NOV005, and NOV006 after 4 weeks at 40° C.

TABLE 3 High molecular weight species (BMW) formation % HMW (Absolutevalue) Concentration 4 weeks at Molecule (mg/mL) Formulation T_(o) 40°C. NOV004 150 20 mM Hist/HC1, n.a n.a 220 mM Sucrose, 0.04% PS2O, pH 5.520 mM Hist/HC1, 1.2 64 220 mM sucrose, 0.04% PS20, pH 6.5 NOV005 150 20mM Hist/HC1, 0.2 0.8 220 mM Sucrose, 0.04% PS2O, pH 5.5 20 mM Hist/HC1,0.2 0.6 220 mM sucrose, 0.04% PS20, pH 6.5 NOV006 150 20 mM Hist/HC1,<LOQ 0.7 220 mM Sucrose, 0.04% PS2O, pH 5.5 20 mM Hist/HC1, <LOQ 0.6 220mM sucrose, 0.04% PS20, pH 6.5 LOQ = limit of quantification

The differences in sequences of NOV005 and NOV006, relative to NOV004,conferred significant improvement in the formation of HMW upon storage.Both NOV005 and NOV006 exhibited approximately less than 1% HMWformation at 40° C. for 4 week, while NOV004 exhibited much higher HMWformation, approximately 64% HMW formation at 40° C. for 4 week.

Sequence alignment of the VH and VL of NOV005 and NOV004 is providedbelow:

VH Identity: 79.2 Similarity: 88.3% NOV005 1QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYTWHWIRQHPGKGLEWIG 50:|||.|||.|||||..:|.|:|.||||||||||||||:||.|||||||:. NOV004 1EVQLVESGGGLVKPGGSLRLSCAVSGYSITSGYTWHWVRQAPGKGLEWLS 50 NOV005 51YIHYSVYTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARRT 100|||||||||||||:|.|.||||||:||.|.|:::|:.|.||||||||||| NOV004 51YIHYSVYTNYNPSVKGRFTISRDTAKNSFYLQMNSLRAEDTAVYYCARRT 100 NOV005 101TSLERYFDVWGQGTLVTVSS 120 |||||||||||||||||||| NOV004 101TSLERYFDVWGQGTLVTVSS 120 VL Identity: 99.1 Similarity: 100.0% NOV005 1DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYY 50|||||||||||||||||||||||||||||||||||||||||||||||||| NOV004 1DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYY 50 NOV005 51TSRLQSGVPSRFSGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTFGQ 100||||||||||||:||||||||||||||||||||||||||||||||||||| NOV004 51TSRLQSGVPSRFTGSGSGADYTFTISSLQPEDIATYFCQQGNTLPYTFGQ 100 NOV005 101GTKLEIK 107 ||||||| NOV004 101 GTKLEIK 107

This data show that NOV005 and NOV006 surprising exhibited minimalaggregation tendencies as indicated by formation of BMW in contrast toNOV004, and suggest that NOV005 and NOV006 exhibit characteristicssuitable, and would be more preferable, for a pharmaceuticalformulation.

Example 5: Study in Obese, Cynomolgus Monkeys

The effects of FGF21 mimetic mAbs, such as NOV004, NOV005, or NOV006, onfood consumption, body weight, and plasma biomarkers in obese cynomolgusmonkeys are studied. Exemplary Protocols:

Five male cynomolgus monkeys are treated with two subcutaneous (s.c.), 1mg/kg doses of FGF21 mimetic mAbs, such as NOV004, NOV005, or NOV006 tobe administered one week apart (study days 0 and 7) and foodconsumption, body weights, and plasma biomarkers are assessed for morethan 100 days post-dose. For each dose, animals are restrained in theirhome cage, blood samples are collected, and then each animal is given asubcutaneous dose of 1 mg/kg FGF21 mimetic mAbs, such as NOV004, NOV005,or NOV006. Food consumption measurements start 1 week before the firstdose and continues through the study. The study diet is weighed prior tofeeding and is divided into two equal portions for each day. Thefollowing morning, remaining diet is collected and weighed. The numberof pellets (1 g each) dropped in the catch pan are counted and are addedto the weight of the remaining food. Daily food consumption iscalculated as the weight of food provided minus food collected. Fruitand vegetable consumption are not measured. Non-fed body weights aremeasured in duplicate three mornings per week (prior to blood collectionor dosing) using the dynamic feature on the scale.

Measurement of Plasma FGF21 Mimetic mAb Concentrations

Human Fc IgG in cynomologus monkey plasma is quantified using an ELISAbased sandwich immunoassay. Anti-human-IgG mouse IgG1, a mousemonoclonal antibody against human IgG, is used as the capture antibody.White, Greiner, 384-well plates are coated with 2 μg/mL anti-human-IgGmouse IgG1 (30 μL/well) and are incubated overnight at room temperature(RT). Coating antibody is aspirated and lx milk blocker (KPL #50-82-01)is added at 90 μL/well for 2 h at RT. The blocking solution is aspiratedand the plates are stored at −80° C. in plate bags with desiccant untilassay. On the day of the assay, plates and reagents are brought to RT.Standards are made by diluting the purified IgG from 4000 to 16 pM incustom casein sample diluent and including a buffer control. Samples arediluted in duplicate 1:50, 1:250, 1:1250, and 1:6250 in the same diluentas standards, and then standards, diluted samples, and controls areadded to the plate for 2 h at RT (working volume for all steps was 30μL/well). Plates are then washed 3 times with a phosphate based washbuffer. Horseradish peroxidase (HRP)-labeled anti-human Fc-gammaantibody is added to the plate for 1 hour at RT, and then the plates arewashed 3 times with a phosphate-based wash buffer. Chemiluminescentsubstrate is added to the plate and the plate is immediately read on aluminescence plate reader.

FGF21 mimetic mAb, e.g., NOV004, NOV005 or NOV006, standards are assayedin triplicate per plate. Diluted plasma samples are assayed induplicate. Unknown samples are interpolated from the IgG standard curve.Curve fitting, back-calculation, % recovery, and interpolation of sampleconcentrations are performed using SoftMax Pro Software v5.4.1. Signalgenerated by the IgG standards was plotted and fit using a 4-parameterlogistical curve-fitting option. Fc concentrations (pM) in plasmasamples are interpolated from the FGF21 mimetic mAb standard curve andmultiplied by dilution factors. The assay lower limit of quantification(LLOQ) and the upper limit of quantification (ULOQ) are determined. LLOQand ULOQ are defined as the lower and upper standard concentration with100% recovery ±20% and CV<20% and then multiplied by the dilutionfactors.

Detection of Anti-Drug Antibodies

Plasma samples are diluted 1:5 in LowCross Buffer (Boca Scientific; BocaRaton, FL; cat no. 100 500). Reaction Mixture is prepared containing 0.6μg/mL of biotin-labeled FGF21 mimetic mAb and 0.6 μg/mL ofdigoxigenin-labeled FGF21 mimetic mAb in LowCross Buffer. Diluted plasma(80 μL) is combined with 160 μL of Reaction Mixture in 96-well U-bottomplates (BD Falcon; Billerica, MA; cat no. 351177). The edges of theplates are sealed with Parafilm and the plates are incubated on ashaking platform at 37° C. for 2 h (150 rpm, protected from light). Analiquot of each mixture (100 μL) is then transferred to duplicate wellsof Streptavidin-coated 96-well plates (Roche; cat no. 11734776001),which are first washed 3 times with wash buffer consisting of 1×PBScontaining 0.05% (v/v) Tween-20 (300 μL per well). The plates are sealedand then incubated at RT on a shaking platform for 1 h 300 rpm,protected from light). Plates are washed 3 times with wash buffer (300μL per well), and then 100 μL of anti-digoxigenin peroxidase POD Fabfragment (Roche; cat no. 11633716001) diluted 1:2500 in LowCross Bufferare added to each well. The plates are sealed, are incubated at RT on ashaking platform for 45 minutes (300 rpm, protected from light), andthen are washed 3 times with wash buffer (300 μL per well). TMB OneComponent HRP Microwell Substrate (Bethyl Laboratories; Montgomery TX;cat no. E102; 100 μL/well) is added to each well and blue color wasdeveloped for 9-10 min., protected from light. The color reaction isstopped by adding 100 μL of 0.18 N H2SO4 to each well, the plates areshaken briefly, and yellow color is measured at OD₄₅₀.

Measurement of Plasma Glucose Concentrations

Plasma glucose concentrations are measured using an Autokit Glucoseassay (Wako Chemicals; Richmond, VA; catalog no. 439-90901). A standardcurve is prepared by diluting the calibrator to 500, 200, 100, 50, 20,and 0 mg/dL standards. Assay reagent (300 μL), pre-warmed to 37° C., isadded to 2 μL of plasma, standards, and control samples in a clear,flat-bottom, 96-well plate (Thermo Scientific; cat no. 269620). Theplate is mixed on a plate shaker for 30 s and then incubated at 37° C.for 5 min. Following a 20 s mix, the plate is read at 505/600 nm using aMolecular Devices SPECTRAmax PLUS 384 (Sunnyvale, CA). Sample glucoseconcentrations are calculated by comparing to the standard curve.

Measurement of Plasma Insulin Concentrations

Measurement of plasma insulin concentrations Plasma insulinconcentrations are determined using the Millipore Human Insulin SpecificRIA Kit (Billerica, MA; cat no. HI-14K) according to the manufacturerinstructions. Appropriate amounts of assay buffer, standards, or dilutedplasma sample are mixed with ¹¹⁵I-insulin and anti-insulin antibody in 5mL, 75×12 mm PP SARSTEDT tubes (catalog no. 55.526). The tubes arevortexed, covered, and incubated for 20 h at RT. After the incubation, 1mL of 4° C. precipitating reagent is added and the tubes are vortexedand incubated for 30 min. at 4° C. All tubes are centrifuged for 30 min(3000 rpm at 4° C.), the supernatants are decanted, and the pellets arecounted on a PerkinElmer WIZARD2 Automatic Gamma Counter (model no.2470; PerkinElmer; Waltham, MA). Insulin concentrations are calculatedby comparing to a standard curve generated using known quantities ofinsulin.

Measurement of Plasma Triglyceride Concentrations

Plasma triglyceride (TG) concentrations are measured using theTriglyceride (GPO) Liquid Reagent set (Pointe Scientific; Canton, MI;cat no. T7532-500). Pre-warmed assay reagent (300 μL, 37° C.) is addedto 5 μL of plasma in a clear, flat-bottom, 96-well plate (Thermo FisherScientific; Tewksbury, MA; cat no. 269620). The plate is mixed on aplate shaker for 30 s and then is placed in an incubator at 37° C. for 5min. Following a 20 s mix, absorbance is measured at 500 nm with aSPECTRAmax PLUS plate reader. TG concentrations are calculated bycomparing to a calibration curve generated using known quantities of aTG standard (Pointe Scientific; cat no. T7531-STD).

Measurement of Plasma Cholesterol Concentrations:

Plasma total cholesterol (TC) is quantified using the Cholesterol(Liquid) Reagent Set, (Pointe Scientific; cat no. C7510-500). Pre-warmedassay reagent (200 μL, 37° C.) is added to 10 μL of plasma in a clear,flat-bottom, 96-well assay plate (Thermo Fisher Scientific; cat no.269620). The plate is mixed on a plate shaker for 30 s and thenincubated at 37° C. for 5 min. Following a 20 s mix, absorbance ismeasured at 500 nm in a SPECTRAmax PLUS plate reader. Cholesterolconcentrations are calculated by comparing to a calibration curvegenerated using known quantities of a cholesterol standard (StanbioLaboratory; Boerne, TX; cat no. 1012-030).

Measurement of Plasma High-Density Lipoprotein CholesterolConcentrations

For determination of high-density lipoprotein (HDL) cholesterolconcentrations, 50 μL of plasma sample is combined with 50 μL ofCholesterol Precipitating Reagent (Wako Chemicals; Richmond, VA; cat no.431-52501) in a 0.5 mL microcentrifuge tube and is vortexed briefly. Thetube is placed at room temperature for 10 min and then is centrifuged at2000×g for 10 min at 4° C. Following centrifugation, approximately halfof the supernatant (containing the HDL cholesterol portion of theoriginal plasma sample) is removed and 10 μL is used for the cholesterolassay described above.

Measurement of Plasma β-Hydroxybutyrate Concentrations

Plasma β-hydroxybutyrate ((β-HIB) concentrations are measured using theβ-Hydroxybutyrate LiquiColor Test kit (Stanbio Laboratory; cat no.2440-058). Assay reagent R1 (215 μL pre-warmed to 37° C.) is added to 20μL of quality control or plasma sample in a clear, flat-bottom, 96-wellplate (Thermo Fisher Scientific; cat no. 269620). The plate is mixed ona plate shaker for 30 s and is then placed in an incubator at 37° C. for5 min. Pre-read absorbance is measured at 505 nm in a SPECTRAmax PLUSplate reader. Assay reagent R2 (35 μL pre-warmed to 37° C.) is added toeach well, and the plate is again mixed on a plate shaker for 30 s andis incubated at 37° C. for 5 min. Following a 20 s mix, final absorbanceis measured at 505 nm from which the pre-read value was subtracted. β-HBconcentrations are calculated by comparing to a calibration curvegenerated using known quantities of a β-HB calibrator (Wako Diagnostics;Richmond, VA; cat no. 412-73791).

Statistical Analyses

Statistical analyses are performed using GraphPad Prism (Version 6.05;GraphPad Software; La Jolla, CA). Food intake data for each animal arenormalized as a percent of baseline (calculated as the mean of days −6to 0) and then group means±standard errors of the mean (SEM) arecalculated; each day is compared to day 0 by nonparametric Friedman'stest with Dunn's multiple comparisons post-test. Body weights arepresented as group means±standard errors of the mean (SEM) calculated aspercent of baseline (calculated as the mean of days −7, −5, −3, and 0).Raw body weight and plasma biomarker data are also analyzed bynonparametric Friedman's test with Dunn's multiple comparisonspost-test. P<0.05 is considered significant.

Example 6: Study in Obese, Cynomolgus Monkeys

To assess the effects of NOV005 in male normoglycemic obese cynomolgusmonkeys, two subcutaneous 1 mg/kg doses of NOV005 (n=5 animals) orvehicle (n=3 animals) were administered one week apart. Interim studyresults are summarized below:

-   -   NOV005 decreased food consumption of standard chow, with a peak        mean reduction of about 60% compared to baseline (FIG. 6A) and a        mean peak weight reduction of about 10% compared to baseline in        NOV005 treated animals (FIG. 6B)    -   Plasma from non-fasted animals was analyzed for changes in        biomarkers of lipid and carbohydrate metabolism:        -   A mean decrease of about 65% was observed for plasma TG            levels in NOV005 treated animals compared to baseline at day            35    -   NOV005 also decreased total cholesterol and insulin compared to        baseline at day 35, but these changes were not statistically        significant    -   Other biomarkers that will be measured at the end of the study        include adiponectin, β-hydroxybutyrate, ApoCIII, HDL-C and        lipoprotein profiles

TABLE 4NOV005 improved plasma biomarker levels in obese cynomolgusmonkeys Biomarker¹ Baseline² Day 35 Mean % Δ³ Triglycerides (mg/dL) 177± 13  47 ± 15 −65 ± 12* Total cholesterol (mg/dL) 108 ± 2  90 ± 9 −17 ±6   Glucose (mg/dL) 64 ± 4 66 ± 9 3 ± 7 Insulin (μU/mL) 212 ± 33 29 ± 7−54 ± 15  Values represent group means ± SEM. ¹Blood was collected fromnon-fasted animals for biomarker measurements. ²Baseline values reflectthe mean of days −7, −3 and 0. ³Percent change was calculated for eachindividual and then averaged for group mean ± SEM. *P < 0.05 vs baselineby nonparametric Friedman test with Dunn's post-test.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

1.-25. (canceled)
 26. A method of treating a metabolic disorder comprising administering to a subject afflicted with the metabolic disorder an effective amount of an antibody that binds to β-klotho, or antigen-binding fragment thereof, comprising a variable heavy chain as set forth in SEQ ID NO: 15, and a variable light chain as set forth in SEQ ID NO: 26 or
 32. 27. The method of claim 26, wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain as set forth in SEQ ID NO: 15 and a variable light chain as set forth in SEQ ID NO:
 26. 28. The method of claim 26, wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain as set forth in SEQ ID NO: 15 and a variable light chain as set forth in SEQ ID NO:
 32. 29. The method of claim 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 28 or
 34. 30. The method of claim 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO:
 28. 31. The method of claim 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO:
 34. 32. The method of claim 26, wherein the subject is afflicted with one of more of the following: obesity, type 1 diabetes mellitus, pancreatitis, dyslipidemia, hypertriglyceridemia, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, and metabolic syndrome.
 33. The method of claim 32, wherein the subject is afflicted with one or more of the following: obesity, diabetes, hypertriglyceridemia, and dyslipidemia.
 34. The method of claim 32, wherein the subject is afflicted with NASH.
 35. The method of claim 32, wherein the subject is afflicted with NAFLD. 